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[18] Avidin- a n d S t r e p t a v i d i n - C o n t a i n i n g P r o b e s
By EDWARD A. BAYERand MEIR WILCHEK The collection of avidin-containing probes (Table I) represents the second major "partner" of the avidin-biotin system. In this chapter, we provide protocols for the preparation of many types of these probes that are not included in the other chapters in the book. Hence, in addition to describing the synthesis of avidin-containing probes commonly in use in our laboratory, we include preparations by others which, we feel, fill a void. As already mentioned in the introductory chapter [2], there are two general approaches in preparing a desired avidin-containing probe: (1) direct (covalent) coupling between the probe and avidin and (2) complex formation between native avidin and a biotinylated probe. Of course, the latter approach includes both the sequential (stepwise) approach and the preparation of preformed complexes. Derivatized Avidins
Fluorescein-Derivatized Avidin In theory, avidin is a better choice than streptavidin for derivatization via amino groups (e.g., for a fluorescent probe). The high number of free lysines provides a good base for extensive derivatization, and the pI of the resultant avidin derivative is reduced to an acceptable level vis-a-vis nonspecific binding. Nonglycosylated avidin is an even better choice, since the lack of sugars removes the other major source of errant binding.
Reagents Avidin (Belovo Soc. Coop., Bastogne, Belgium; or STC Laboratories, Winnipeg, Manitoba), 10 mg/ml, or nonglycosylated avidin, l dissolved in 10 mM phosphate buffer (pH 7.4) Fluorescein isothiocyanate (FITC), 2 isomer I (Sigma Chemical Co., St. Louis, MO), 2.5 mg/ml in 0.5 M sodium bicarbonate buffer (pH 9.5) Y. Hiller, E. A. Bayer, and M. Wilchek, this volume [6]. 2 We use 25/~g fluorescein isothiocyanate per milligram of avidin, which is equivalent to about 20 mol F I T C / m o l e avidin.
METHODS IN ENZYMOLOGY,VOL. 184
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
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TABLE I COMMONLY USED AVIDIN-CONTAININGPROBESa Probes (conjugates) Enzymes
Radiolabels Fluorescent agents Chemiluminescent agents Chromaphores Heavy metals Colloidal gold Ferritin Hemocyanin Phages Macromolecular carders
Liposomes Solid supports
a
Applications Immunoassay, diagnostics, blotting, affinity cytochemistry [light microscopy (LM), electron microscopy (EM)], affinity perturbation Immunoassay, cytochemistry, cytological probe Affinity cytochemistry (fluorescence microscopy), flow cytometry, immunoassay, diagnostics Immunoassay, diagnostics Immunoassay, diagnostics Affinity cytochemistry (LM, EM), immunoassay, blotting Affinity cytochemistry (LM, EM), immunoassay, blotting Affinity cytochemistry (EM), macromolecular cartier Affinity cytochemistry (EM), macromolecular carrier Affinity cytochemistry (EM), affinity targeting, diagnostics Cross-linking studies, signal amplification, affinity targeting, drug delivery, cytological probe, affinity fusion, affinity perturbation, affinity partitioning Affinity fusion, drug delivery, affinity targeting, signal amplification Affinity chromatography, immobilization, selective retrieval, selective elimination
As described in [2] in this volume, the same probes can also be derivatized with biotin, and native (underivatized) avidin can be used (either by sequential application or in preformed complexes) to cross-link the biotinylated probe with a biotinylated binder.
Sephadex G-50 column (Pharmacia, Uppsala, Sweden), 10-20 ml bed volume DEAE-cellulose column (DE-52, Whatman), 10 ml bed volume 10 mM Sodium phosphate buffer (pH 8.0) (PB) 0.1 M NaC1 in PB 0.25 M NaC1 in PB Procedure. The avidin solution (1 ml) is mixed with 0.1 ml FITC, and the reaction is carried out overnight at 4° . The conjugate is applied to the Sephadex G-50 column (preequilibrated with PB), and the first peak is collected. The sample is loaded on a DEAE-cellulose column, preequilibrated with PB, and washed with the same buffer. Unlabeled avidin can be collected from the effluent. In order to elute FITC-labeled avidin, rinse the column with 0.1 M NaCI in PB. Collect the labeled peak, and then elute the column again, this time with 0.25 M NaC1 in PB. The peak fractions are pooled separately and stored at -20 ° . The average number of fluorescein (F) molecules coupled to avidin
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(Av) can be calculated from the following equation: F/Av
=
0.3(A495) A280 - 0.35(A495)
The initial FITC-labeled avidin peak was found to contain an average of 6 fluorescein groups per avidin tetramer; the second labeled peak contained an average of about 9 fluorescein groups per avidin molecule. Tritiated A vidin Tritiated forms of the avidins can be prepared at relatively high specific radioactivity as a stable radiolabeled alternative to iodinated derivatives. Reagents Avidin (Belovo or STC Laboratories), nonglycosylated avidin, ~ or streptavidin, 3 1 mg/ml in 50 mM NaHCO3 N-Succinimidyl [2,3-3H]propionate 4 (Radiochemical Center, Amersham, Buckinghamshire, England), 120 Ci/mmol, 1 mCi Dimethylformamide Sephadex G-50 column, 10 ml bed volume Phosphate-buffered saline (pH 7.4) (PBS) Procedure. The solution (100/~1) of the desired protein 5 is introduced to the vial containing the dimethylformamide solution of the tritiated reagent. After I hr at room temperature, the reaction mixture is applied to the Sephadex G-50 column (preequilibrated with PBS), and the first peak is collected. The peak fractions are pooled and stored in aliquots at - 2 0 °. Comments. This procedure routinely results in stable tritiated derivatives of about 107 cpm//~g for avidin and about 3 × 106 cpm//zg for streptavidin. The ratio of egg-white avidin to reagent used in this protocol represents a compromise for achieving optimal levels of both biotin-binding activity and radiolabeling. The given ratio has the added advantage of reducing the pI of the radiolabeled derivative; if nonglycosylated avidin is used, the resultant conjugate is similar to streptavidin in its nonspecific binding properties. 3 L. Chaiet and F. J. Wolf, Arch. Biochem. Biophys. 1116, 1 (1964). 4 The reagent obtained from Amersham is dissolved in 1 ml of toluene. In order to carry out the radiolabeling of avidin, the toluene is evaporated in the original vial. The residue is then redissolved in 10/zl dimethylformamide. 5 Final concentrations of 0.3 mg/ml for avidin or nonglycosylated avidin can be used in order to enhance the level of tritiation of the product; we usually perform the tritiation of streptavidin at 1 mg/ml.
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Avidin-Conjugated Proteins
Ferritin-Aoidin Conjugates There are many methods available for the conjugation of two proteins. Several approaches have been taken to produce viable ferritin-avidin conjugates, including glutaraldehyde coupling, 6 cross-linking of bromoacetylated ferritin with thiolylated avidin, 7 and reductive alkylation, 8 The latter procedure consists of periodate-induced oxidation of the oligosaccharide residues on avidin; the aldehydes produced react with amino groups of the ferritin, and the resultant Schiff base is further reduced to a stable bond with borohydride.
Reagents Avidin (Belovo or STC Laboratories), 9 15 mg dissolved in 5 ml acetate-buffered saline (pH 4.5) Ferritin, 2 times recrystallized (Sigma), 100 mg in 0.1 M NaCI 1 ml solution Sodium metaperiodate (Merck, Darmstadt, Federal Republic of Germany) Sodium borohydride (Merck) Acetate-buffered saline [50 m M sodium acetate buffer (pH 4.5) containing 0.15 M NaCI] 0.1 M Borate-buffered saline (pH 8.5) PBS (pH 7.4) Iminobiotin-Sepharose column ~° Procedure. The solutions of avidin and ferritin are mixed and periodate (0.66 ml, 0.1 M solution) is added to a final concentration of 10 raM. The solution is stirred for 30 min on ice, dialyzed against acetatebuffered saline for 6 hr at 4°, and immediately dialyzed overnight at 4° against borate-buffered saline. A fresh solution of sodium borohydride (10 mg/ml in 10 m M NaOH) is prepared, and 0.5 ml is added to the ferritinavidin conjugates in an ice bath. After 1 hr, the solution is dialyzed against PBS. The conjugates are washed twice by centrifugation (100,000 g, 3 hr 6 H. Heitzmann and F. M. Richards, Proc. Natl. Acad. Sci. U.S.A. 71, 3537 (1974). L. Angerer, N. Davidson, W. Murphy, D. Lynch, and G. Attardi, Cell 9, 81 (1976). s E. A. Bayer, E. Skutelsky, D. Wynne, and M. Wilchek, J. Histochem. Cytochern. 24, 933 (1976). 9 STC avidin has been shown by us to contain the full complement of oligosaccharides and is thus similar in its molecular properties to that obtained from Sigma (the former is about 10 times cheaper). Belovo avidin is even cheaper than STC avidin, but its sugar residues are partially degraded. 10 E. A. Bayer, H. Ben-Hur, and M. Wilchek, this volume [8].
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at 4 °) in order to remove free (unconjugated) avidin molecules, and the pellet is resuspended to 1 mg/ml ferritin. The avidin-containing species are separated from the free ferritin by affinity chromatography on an iminobiotin-Sepharose column. 1° The conjugates are passed through a sterile Millipore filter (HA 0.2/zm) and stored in aliquots under sterile conditions at 4 °. Comments. The procedure given here results in a high yield of unitpaired conjugates, which are particularly suitable for precise ultrastructural localization of biotinylated sites associated with subcellular structures. The procedure can, of course, be used to conjugate avidin to other proteins. In cases where the given protein is sensitive to periodate, avidin alone can be preoxidized, the periodate removed by dialysis or gel filtration, and the oxidized avidin reacted further with the desired protein. Owing to the directionality of the above-described chemistry, this procedure is applicable to cases where relatively small conjugates are required.
Avidin- Toxin Conjugates Although we have not included in this volume the use of the avidinbiotin system for therapeutic purposes, the use of avidin-drug or avidintoxin conjugates has recently been seriously considered in studies of cancer therapy. This basic approach is also being used for imaging purposes using conjugates of avidin with radioactive metals. In most of these cases, a biotinylated antibody is first delivered to the target (tissue or organ), and then the avidin-associated probe is introduced. The probe can be either covalently attached to avidin or complexed via a biotin bridge. In some cases, after the biotinylated antibody is interacted with the system, native (underivatized) avidin is delivered, followed sequentially by the biotinylated toxin or radioactive metal. In this chapter, we describe only the preparation of an avidin-toxin conjugate (avidin-ricin A chain). In this case, an active disulfide-containing avidin derivative is interacted with the A chain of ricin (which contains a free sulfhydryl group), thus forming a mixed disulfide conjugate. 11
Reagents Avidin (Belovo or STC Laboratories), nonglycosylated avidin, ~ or streptavidin, 3,1° 10 mg in 4 ml of PBS Ricin A chain (Sigma), 5 mg Acetate-buffered saline (pH 4.5) PBS (pH 7.4) II N. Hashimoto, K. Takatsu, Y. Masuho, K. Kishida, T. Hara, and T. Hamaoka, J. Imrnunol. 132, 129 (1984); K. A. Krolick, J. W. Uhr, and E. S. Vitetta, this series, Vo|. 93 [21].
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N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP, Pharmacia), 10 mg/ml in dimethylformamide Sephacryl S-200 column (1.6 x 88 cm, Pharmacia), equilibrated with PBS Procedure. An aliquot (30 tzl) of SPDP is added to the solution of avidin, and the reaction is allowed to proceed at room temperature for 30 min. The reaction mixture is dialyzed against acetate-buffered saline for 3 hr in order to remove extraneous reagent. Ricin A chain is dissolved in 2.5 ml of acetate-buffered saline, and the solution is dialyzed against the same buffer for 1 hr. The dialyzed solutions of SPDP-modified avidin and ricin A chain are mixed, and the reaction is allowed to proceed overnight at room temperature. The mixture is dialyzed against PBS for 3 hr, and the avidin-ricin A chain conjugates are separated from unreacted avidin and toxin by gel filtration on a Sephacryl S-200 column. The conjugates are passed through a sterile Millipore filter (HA 0.2/~m) and stored in aliquots under sterile conditions at 4 °. Comments. There are many other methods that can be used to couple a toxin to a given carrier. However, in order to obtain an active avidintoxin conjugate, the bond connecting the two should be subject to disassociation. The preferred method is to form a disulfide linkage that would be susceptible to reducing conditions which characterize the intracellular matrix. Internalization is very important for toxin action; the use of cocomplexes containing biotinylated antibody and biotinylated toxin connected via a native (underivatized) avidin bridge may enhance cross-linking on the cell surface and result in internalization of the complexed components into the target cells. The native avidin may also serve as a scavenger to remove rapidly from the circulation free residual toxin and antibody molecules that fail to react with target cells.
Avidin-Penicillinase Conjugates One of the earliest examples of a reagent to be used for conjugating proteins is glutaraldehyde. 12Although this is an example of a homobifunctional reagent and the directionality of the cross-linking cannot be controlled, glutaraldehyde is still a very popular reagent for covalent coupling of proteins. The reason for this (over and above historical precedence and its use out of habit) is simply that the procedure is easy and yields reasonably good results. As an example of this mode of cross-linking, we detail below the coupling of avidin to penicillinase, an interesting enzyme which should find extensive use in immunoassays. One of the possible substrate for12 S. Avrameas,
lmmunochemistry 6, 43 (1969).
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GENERALMETHODOLOGY 400
. . . .
I
Void
. . . .
I
Fer
[18] . . . . Av
200
PCase
300
O
150
200
i00
0 20
I
j
,
-
i00
-
50
,
0
30 Fraction
c~
40
50
number
FIG. 1. Gel filtration of avidin-penicillinase conjugates. Following glutaraldehyde crosslinking of avidin and penicillinase, the reaction mixture was dialyzed and applied to a Sephacryl S-300 column (1.6 x 76 cm) preequilibrated with PBS. The resultant fractions (2 ml) were analyzed for protein content (0) and enzymatic activity (©). Protein content is given as/,~g/fraction, and penicillinase activity represents the reciprocal of time required for the disappearance of color in the substrate solution (hr-l). Void, Fer, Av, and PCase represent the positions of eluted standards for the void volume, ferritin, avidin, and penicillinase, respectively.
mats (presented below) results in the disappearance (as opposed to production) of color, which can be particularly useful in hybridoma technology, pregnancy tests, or any assay where a yes/no answer is required. Reagents
Avidin (Belovo or STC Laboratories), nonglycosylated avidinJ or streptavidin, 3,1° 2 mg ( - 3 0 nmol) dissolved in 0.4 ml PBS Penicillinase from Bacillus cereus (Sigma, -2000 units/mg), 0.5 mg protein ( - 2 0 nmol enzyme) dissolved in 0.1 ml PBS PBS (pH 7.4) Glutaraldehyde solution: 12.4 /zl of an 8% (v/v) solution (Polysciences, Inc., Warrington, PA) is brought to 1.5 ml with PBS Sephacryl S-300 column (1.6 × 76 cm) Substrate solution13:3 ml of 0.5% gelatin (w/v in distilled water), 0.5 13 The substrate solution for penicillinase is prepared immediately before use.
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181
ml of 25 m M iodine in 0.125 M potassium iodide, 0.1 ml of 1% (w/v) soluble starch, and 1 ml of benzylpenicillin [3 mg/ml in 0.1 M phosphate buffer (pH 7)] Procedure. The solutions of avidin and penicillinase are mixed, and the glutaraldehyde solution is added (0.05% final concentration). After 2 hr at room temperature, the slight precipitate that forms is centrifuged, and the supernatant fluids are dialyzed against PBS. The contents of the dialysis tubing are subjected to gel filtration on a Sephacryl S-300 column, and the void-volume fractions are pooled, passed through a sterile Millipore filter (HA 0.2/zm), and stored in aliquots under sterile conditions at 4 °. Comments. Glutaraldehyde-mediated cross-linking of two protein species usually results in the formation of high molecular weight multimers of the two. This is indeed the case with avidin and penicillinase (Fig. 1). Using the conjugation procedure described above, very little free (unconjugated) avidin and penicillinase remain; the largest fraction appears in the void volume (> 1,000,000), and a trail of lower molecular weight multimers can also be recovered. The void-volume fractions (25-27 in Fig. 1) are pooled, and the preparation was found to exhibit high levels of both biotin-binding and penicillinase activities. The preparation is appropriate for immunoassay studies. 14Penicillinase can also be biotinylated and used in conjunction with underivatized avidin to form complexes, similar to those described below. Avidin-Protein Complexes
Avidin and Streptavidin Complexes with Biotinylated Enzymes Preformed complexes between avidin and biotinylated enzymes provide an alternative to classic covalent cross-linking of two proteins to form the corresponding conjugate. The major advantages in using conjugates is that the resultant signal is often enhanced. Moreover, the stock solutions of avidin and many of the biotinylated enzymes can be stored effectively with essentially no deleterious effect on their performance. The preparation of such complexes is convenient, reliable, and reproducible. In forming complexes, a compromise must be reached between the amount of (biotinylated) enzyme which reacts with avidin and the average number of free (available) biotin-binding sites which remain on the avidin molecule. The former is needed to provide a good signal, and the latter is essential for the primary interaction with the desired biotinylated binder 14 R. H. Yolken, this v o l u m e [61].
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(e.g., antibody). The most effective ratio of avidin or streptavidin for complex formation with a given biotinylated enzyme is determined empirically by optimization experiments. In a convenient procedure we designed for this purpose, biotinylated bovine serum albumin is used as a model target system for determining the required conditions for efficient complex formation. Biotinylated alkaline phosphatase (B-AP) is the test enzyme in this example.
Reagents Avidin (Belovo or STC Laboratories), nonglycosylated avidin, l or streptavidin, 3,1° 1 mg/ml in PBS 15 B - A P , 16 500 units/ml in 0.15 M NaC115 PBS (pH 7.4) Bovine serum albumin (BSA), 2% (w/v) in PBS Lysozyme, 2% (w/v) in PBS Biotinylated B S A , 16 1 mg/ml in PBS Substrate solution: 10 mg naphthol AS-MX phosphate (free acid) is dissolved in 200 ~1 of dimethylformamide, and the solution is mixed with a solution containing 30 mg Fast Red dissolved in 100 ml of 0.1 M Tris-HCl (pH 8.4) Optimization Procedure. Complexes consisting of different ratios of avidin (or streptavidin) and B - A P are prepared in the following manner. The stock solution of avidin is diluted to 1, 3, 10, and 30/xg/ml (4 ml total solution each).~7 Similarly, the stock solution of B - A P is diluted (e.g., 1, 2, 5, and 10 units/ml, 4 ml total). ~8A 1-ml sample of each concentration of avidin is combined with an equivalent volume of one of the B - A P solutions in matrix fashion, yielding 16 separate solutions of varying ratios and quantities of avidin to B-AP. The solutions are allowed to stand for about 30 min at room temperature before being incubated with dot blots containing the target material. The biotinylated target protein is applied to dot blots in the following manner. Samples (1 /xl) containing serial dilutions of biotinylated BSA, are applied onto 16 dot blots. 19 The strips are rinsed with PBS, treated with quenching solution, z° and rinsed again, and each blot is incubated in one of the above-described preformed complex preparations. After 30 15 Stock solutions o f avidin (streptavidin) and B - A P are stored in aliquots at - 2 0 °. The stock solutions are u s e d no m o r e t h a n twice (thawed, refrozen, and rethawed). 16 E. A. B a y e r and M. Wilchek, this v o l u m e [14]. 17 L y s o z y m e solution is u s e d as a diluent for avidin, and B S A is u s e d for streptavidin. ~8 B S A is u s e d as a diluent for B - A P . ~9 E. A. Bayer, H. B e n - H u r , and M. Wilchek, this volume [48]. 2o B S A is u s e d as q u e n c h e r for streptavidin-based complexes, and a 1 : 1 mixture of B S A and l y s o z y m e is u s e d for avidin-based complexes.
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B-AP (Ulml) 2
I
5
I0
I J
i i
•
i~l
f
hill
o •
0 l, e
i
0 0
Q •
0
0
0 (j
0 oe
III
qllJ e
tqDO
I
Q' • e
0
q~ i '
30 t
FIG. 2. Dot-blot optimization of complex formation between avidin and B-AP. Onto each nitrocellulose strip, 1-/~1 s a m p l e s containing serial dilutions (top row: 1000, 300, 100, 30, 10, and 3 ng//xl; b o t t o m row: 1, 0.3, 0.1, etc.) of biotinylated B S A were applied. The strips were rinsed, q u e n c h e d , rinsed again, and incubated in solutions containing the different c o m p l e x e s . T h e designated a m o u n t s indicate the initial concentrations of either B - A P or avidin solutions that were c o m b i n e d 1 : 1 and applied to the corresponding nitrocellulose strip. Following incubation, the strips were rinsed, and substrate solution was added.
min, the strips are rinsed again with PBS, and substrate solution is added. Bands usually appear within 30 min; at the desired amplification, the reaction is terminated by washing the strips with tap water. Standard Procedure. Once the system has been optimized for a given biotinylated enzyme preparation, the procedure for complex formation can be standardized using a given ratio of components. Using the B - A P prepared in our laboratory, ~6we found the following protocol to result in optimal levels of complexation of the enzyme with avidin or streptavidin. A 100-fold dilution 17,18 of the above stock solutions of both avidin (or streptavidin) and B - A P are combined and used after a 30-min incubation at room temperature. Comments. The optimal ratio for complex formation corresponds to about 2/xg avidin per unit of B - A P (Fig. 2); initial concentrations (before mixing) of 10/~g/ml avidin and 5 units/ml B - A P are generally used. At relatively low avidin-to-B-AP ratios (i.e., 1 /xg/ml and 10 units/ml, respectively), little labeling can be observed, presumably since most of the biotin-binding sites of avidin are involved in complex formation and few free sites are available to combine with the biotinylated target protein. Conversely, at high avidin-to-B-AP ratios (i.e., 30/zg/ml and 1 unit/ml, respectively), the labeling pattern is also impaired, apparently because
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extraneous uncomplexed avidin molecules compete with the complexes, thereby reducing the amount of enzyme available for detection. It should be noted that the concentration of complexes introduced into the system should be regulated in order to reduce the level of nonspecific binding. Specifically, high background levels are observed at B-AP concentrations higher than 10 units/ml, and such conditions should be avoided. The optimal ratio for streptavidin-containing complexes is identical to that of avidin. The use of streptavidin usually results in much lower levels of either nonspecific binding or background, and it is generally recommended when available. For new batches of biotinyl enzyme, such optimization experiments should always be performed. Stored samples of reagents should also be subjected periodically to such experiments; the complexes themselves are not stable for long periods of time and cannot be stored.
Avidin and Streptauidin Hydrazides Hydrazide derivatives of avidin and streptavidin have been shown to be effective for selectively labeling glycoconjugates on blots. 19,2~The avidin hydrazide is combined with a biotinylated enzyme at an optimized ratio to form an amplified enzyme-hydrazide preparation, which can be used to label periodate-oxidized or galactose oxidase-treated saccharides.
Reagents Avidin (Belovo or STC Laboratories), nonglycosylated avidin, 1 or streptavidin, 3,1° 50 mg/ml Adipic acid dihydrazide (Sigma), 160 mg Water-soluble carbodiimide, 22 160 mg Distilled water 5 N HC1 PBS (pH 7.4) Procedure. The dihydrazide is dissolved by heating in 5 ml of distilled water, and the pH is adjusted to 5 with HC1. The solution is added to solid avidin or streptavidin (50 mg of the desired protein). The water-soluble carbodiimide is added in solid form, and the reaction is allowed to proceed for 3 hr at room temperature with periodic 30-min adjustments of the pH to 5. The avidin hydrazide is dialyzed exhaustively against PBS. The product is diluted to 50 ml with PBS, passed through a sterile Millipore filter (HA 0.2/xm), and stored in 1-ml aliquots under sterile conditions at 4°. Both avidin and streptavidin hydrazide are stable for years under these conditions. 21 E. A. Bayer, H. Ben-Hur, and M. Wilchek, Anal. Biochem. 161, 123 (1987). 22 l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Sigma).
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185
Complexes of Biotinylated Enzymes with Avidin (or Streptavidin) Hydrazides. To a solution of avidin hydrazide or streptavidin hydrazide (15 /xg/ml, dissolved in 2% w/v lysozyme in PBS) is mixed an equal volume of biotinyl alkaline phosphatase 15,~6(7.5/xg/ml). 23 Comments. The optimal ratio of hydrazide probe to biotinyl enzyme for complex formation is determined by dot-blot optimization experiments similar to that described above for avidin-enzyme complexes, except that the target molecule consists of a suitable glycoconjugate (e.g., fetuin) which is oxidized by enzymatic or chemical means.19 The ratio of reagents is based on the results of such an experiment. As mentioned above, for new batches of biotinyl enzyme and avidin (or streptavidin) hydrazide, such optimization experiments should always be performed. Stored samples of reagents should also be subjected periodically to such experiments. The solution is applied to blots about 30 min after initiation of complex formation; the complexes themselves are not stable for long periods of time and cannot be stored. Derivatization of a protein via its carboxyl group results in increased pI values; a neutral protein such as streptavidin becomes basic, and a basic protein such as avidin becomes even more basic. This phenomenon generates high levels of nonspecific binding which can easily be countered by quenching with appropriate reagents. 23 Thus, in lieu of using albumin (a negatively charged protein) as a quencher, we employ lysozyme (the pI of which is even higher than that of avidin), zl
Immobilized Avidins Avidin can be immobilized to solid matrices in many different ways. The classic method involving CNBr activation of Sepharose has been described explicitly in the past. 24 In this section, we present two alternative methods for immobilization of avidin to Sepharose.
Cyano-Transfer Activation of Sepharose In the first method, the use of the extremely hazardous CNBr for activation is precluded by using a cyano-transfer derivative or intermediate. 25 23 For complexes containing avidin hydrazide, 2% lysozyme is used as a diluent; for complexes containing streptavidin hydrazide, the diluent consists of 2% BSA. 24 E. A. Bayer, E. Skutelsky, and M. Wilchek, this series, Vol. 62 [55]; E. A. Bayer and M. Wilchek, Methods Biochem. Anal. 26, 1 (1980); A. Bodanszky and M. Bodanszky, Experientia 26, 237 (1970). 25 M. Wilchek, T. Miron, and J. Kohn, this series, Vol. 104 [I].
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Reagents Avidin (Belovo or STC Laboratories), nonglycosylated avidin, l or streptavidin, 3,~° 200 mg in 100 ml of 0.1 M sodium bicarbonate Sepharose 4B or CL-4B, 50 ml of swollen gel N - C y a n o t r i e t h y l a m m o n i u m tetrafluoroborate (CTEA), 540 mg, or 1cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP), 225 mg z6 (Sigma) 0.2 M Triethylamine (aqueous solution) Acetone Distilled water Washing medium: acetone : 0.I N HCI (1:1, v/v) Storage medium: acetone : dioxane : water (60 : 35 : 5, v/v) Coupling medium: 0.1 M sodium bicarbonate (pH 8.5) PBS (plus 0.1% sodium azide, p H 7.4) Procedure. The Sepharose is washed with water, 30% acetone, and 60% acetone, successively, and resuspended in 10 ml of 60% acetone. The suspension is cooled to 0 °, and the desired cyano-transfer reagent is added. The triethylamine solution (5.4 ml for the CTEA-activated resin or 1.8 ml for the CDAP-activated resin) is then added dropwise with vigorous stirring. After 2 min, the reaction mixture is transferred to 100 ml of ice-cold washing medium. The resin can be maintained in this manner for about 1 hour without loss of active groups. 27 The activated resin is washed with large volumes of cold water, followed by a rapid wash with coupling medium. The solution of avidin is mixed with the resin, and the interaction is carried out for 1 hr at room temperature and overnight at 4 ° . The resin is washed with coupling buffer and PBS, 28 and stored in PBS. Comments. The above procedure results in about 80-90% coupling, z8 yielding about 3.5 mg avidin/ml swollen gel. The end products of the reaction are identical to those of the C N B r activation procedure, and a certain amount of leakage of avidin may thus be expected. 25 Nevertheless, these reagents are nonvolatile solid cyanoderivatives which can be stored and handled without use of a fume hood; they should therefore be used in place o f C N B r since they are more convenient, more efficient and, of course, safer. 26Both reagents can be obtained from Sigma. 27For prolonged storage of cyano-transfer-activated Sepharose, the resin is washed extensively with storage medium and maintained at -20 °. When required, the activated resin is reswollen for 5 min in cold washing medium and washed according to the procedure for immediate coupling. 2s In the case of CTEA-activated resins, the A2s0of the washes can be followed in order to determine the extent of coupling.
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p-Nitrophenyl Chloroformate-Activated Sepharose One of the major problems with CNBr (or cyano-transfer) activation of Sepharose is the observed leakage of ligand owing to the instability of some of the bonds formed. Alternative procedures have therefore been developed which provide improved chemical attachment of proteins and other ligands. 25 One of the most effective is the use of chloroformates that result in activated carbonate groups on the resin. Subsequent interaction with amines yields stable and uncharged carbamate (urethane) derivatives.
Reagents Avidin (Belovo or STC Laboratories), nonglycosylated avidin, l or streptavidin, 3,~° 200 mg in 100 ml of 0.1 M sodium bicarbonate Sepharose CL-4B (Pharmacia), 50 ml p-Nitrophenyl chloroformate (Aldrich Chemical Company, Milwaukee, WI), 3 g 4-Dimethylaminopyridine (Sigma), 2.1 g Acetone (absolute) 2-Propanol Distilled water Coupling medium: 0. I M sodium bicarbonate (pH 8.5) PBS (plus 0.1% sodium azide, pH 7.4) Procedure. The activation procedure is carried out as described elsewhere in this volume, l° The solution of avidin is then mixed with the washed, activated resin; the interaction is carried out for 1 hr at room temperature and overnight at 4 °. The resin is washed successively with water, 0.2 M acetic acid, water, and (rapidly) with 10 mM NaOH. The resin is then washed exhaustively with distilled water, followed by coupling buffer and PBS. The immobilized avidin is stored in PBS. Comments. Using the above procedure, about 3 mg avidin can be coupled per milliliter of Sepharose. The covalent bonds formed are very stable, and leakage is negligible. In other respects, the performance of such columns is equivalent to that of CNBr-activated resins.
GENERALMETHODOLOGY
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[18] Avidin- a n d S t r e p t a v i d i n - C o n t a i n i n g P r o b e s
By EDWARD A. BAYERand MEIR WILCHEK The collection of avidin-containing probes (Table I) represents the second major "partner" of the avidin-biotin system. In this chapter, we provide protocols for the preparation of many types of these probes that are not included in the other chapters in the book. Hence, in addition to describing the synthesis of avidin-containing probes commonly in use in our laboratory, we include preparations by others which, we feel, fill a void. As already mentioned in the introductory chapter [2], there are two general approaches in preparing a desired avidin-containing probe: (1) direct (covalent) coupling between the probe and avidin and (2) complex formation between native avidin and a biotinylated probe. Of course, the latter approach includes both the sequential (stepwise) approach and the preparation of preformed complexes. Derivatized Avidins
Fluorescein-Derivatized Avidin In theory, avidin is a better choice than streptavidin for derivatization via amino groups (e.g., for a fluorescent probe). The high number of free lysines provides a good base for extensive derivatization, and the pI of the resultant avidin derivative is reduced to an acceptable level vis-a-vis nonspecific binding. Nonglycosylated avidin is an even better choice, since the lack of sugars removes the other major source of errant binding.
Reagents Avidin (Belovo Soc. Coop., Bastogne, Belgium; or STC Laboratories, Winnipeg, Manitoba), 10 mg/ml, or nonglycosylated avidin, l dissolved in 10 mM phosphate buffer (pH 7.4) Fluorescein isothiocyanate (FITC), 2 isomer I (Sigma Chemical Co., St. Louis, MO), 2.5 mg/ml in 0.5 M sodium bicarbonate buffer (pH 9.5) Y. Hiller, E. A. Bayer, and M. Wilchek, this volume [6]. 2 We use 25/~g fluorescein isothiocyanate per milligram of avidin, which is equivalent to about 20 mol F I T C / m o l e avidin.
METHODS IN ENZYMOLOGY,VOL. 184
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
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TABLE I COMMONLY USED AVIDIN-CONTAININGPROBESa Probes (conjugates) Enzymes
Radiolabels Fluorescent agents Chemiluminescent agents Chromaphores Heavy metals Colloidal gold Ferritin Hemocyanin Phages Macromolecular carders
Liposomes Solid supports
a
Applications Immunoassay, diagnostics, blotting, affinity cytochemistry [light microscopy (LM), electron microscopy (EM)], affinity perturbation Immunoassay, cytochemistry, cytological probe Affinity cytochemistry (fluorescence microscopy), flow cytometry, immunoassay, diagnostics Immunoassay, diagnostics Immunoassay, diagnostics Affinity cytochemistry (LM, EM), immunoassay, blotting Affinity cytochemistry (LM, EM), immunoassay, blotting Affinity cytochemistry (EM), macromolecular cartier Affinity cytochemistry (EM), macromolecular carrier Affinity cytochemistry (EM), affinity targeting, diagnostics Cross-linking studies, signal amplification, affinity targeting, drug delivery, cytological probe, affinity fusion, affinity perturbation, affinity partitioning Affinity fusion, drug delivery, affinity targeting, signal amplification Affinity chromatography, immobilization, selective retrieval, selective elimination
As described in [2] in this volume, the same probes can also be derivatized with biotin, and native (underivatized) avidin can be used (either by sequential application or in preformed complexes) to cross-link the biotinylated probe with a biotinylated binder.
Sephadex G-50 column (Pharmacia, Uppsala, Sweden), 10-20 ml bed volume DEAE-cellulose column (DE-52, Whatman), 10 ml bed volume 10 mM Sodium phosphate buffer (pH 8.0) (PB) 0.1 M NaC1 in PB 0.25 M NaC1 in PB Procedure. The avidin solution (1 ml) is mixed with 0.1 ml FITC, and the reaction is carried out overnight at 4° . The conjugate is applied to the Sephadex G-50 column (preequilibrated with PB), and the first peak is collected. The sample is loaded on a DEAE-cellulose column, preequilibrated with PB, and washed with the same buffer. Unlabeled avidin can be collected from the effluent. In order to elute FITC-labeled avidin, rinse the column with 0.1 M NaCI in PB. Collect the labeled peak, and then elute the column again, this time with 0.25 M NaC1 in PB. The peak fractions are pooled separately and stored at -20 ° . The average number of fluorescein (F) molecules coupled to avidin
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(Av) can be calculated from the following equation: F/Av
=
0.3(A495) A280 - 0.35(A495)
The initial FITC-labeled avidin peak was found to contain an average of 6 fluorescein groups per avidin tetramer; the second labeled peak contained an average of about 9 fluorescein groups per avidin molecule. Tritiated A vidin Tritiated forms of the avidins can be prepared at relatively high specific radioactivity as a stable radiolabeled alternative to iodinated derivatives. Reagents Avidin (Belovo or STC Laboratories), nonglycosylated avidin, ~ or streptavidin, 3 1 mg/ml in 50 mM NaHCO3 N-Succinimidyl [2,3-3H]propionate 4 (Radiochemical Center, Amersham, Buckinghamshire, England), 120 Ci/mmol, 1 mCi Dimethylformamide Sephadex G-50 column, 10 ml bed volume Phosphate-buffered saline (pH 7.4) (PBS) Procedure. The solution (100/~1) of the desired protein 5 is introduced to the vial containing the dimethylformamide solution of the tritiated reagent. After I hr at room temperature, the reaction mixture is applied to the Sephadex G-50 column (preequilibrated with PBS), and the first peak is collected. The peak fractions are pooled and stored in aliquots at - 2 0 °. Comments. This procedure routinely results in stable tritiated derivatives of about 107 cpm//~g for avidin and about 3 × 106 cpm//zg for streptavidin. The ratio of egg-white avidin to reagent used in this protocol represents a compromise for achieving optimal levels of both biotin-binding activity and radiolabeling. The given ratio has the added advantage of reducing the pI of the radiolabeled derivative; if nonglycosylated avidin is used, the resultant conjugate is similar to streptavidin in its nonspecific binding properties. 3 L. Chaiet and F. J. Wolf, Arch. Biochem. Biophys. 1116, 1 (1964). 4 The reagent obtained from Amersham is dissolved in 1 ml of toluene. In order to carry out the radiolabeling of avidin, the toluene is evaporated in the original vial. The residue is then redissolved in 10/zl dimethylformamide. 5 Final concentrations of 0.3 mg/ml for avidin or nonglycosylated avidin can be used in order to enhance the level of tritiation of the product; we usually perform the tritiation of streptavidin at 1 mg/ml.
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Avidin-Conjugated Proteins
Ferritin-Aoidin Conjugates There are many methods available for the conjugation of two proteins. Several approaches have been taken to produce viable ferritin-avidin conjugates, including glutaraldehyde coupling, 6 cross-linking of bromoacetylated ferritin with thiolylated avidin, 7 and reductive alkylation, 8 The latter procedure consists of periodate-induced oxidation of the oligosaccharide residues on avidin; the aldehydes produced react with amino groups of the ferritin, and the resultant Schiff base is further reduced to a stable bond with borohydride.
Reagents Avidin (Belovo or STC Laboratories), 9 15 mg dissolved in 5 ml acetate-buffered saline (pH 4.5) Ferritin, 2 times recrystallized (Sigma), 100 mg in 0.1 M NaCI 1 ml solution Sodium metaperiodate (Merck, Darmstadt, Federal Republic of Germany) Sodium borohydride (Merck) Acetate-buffered saline [50 m M sodium acetate buffer (pH 4.5) containing 0.15 M NaCI] 0.1 M Borate-buffered saline (pH 8.5) PBS (pH 7.4) Iminobiotin-Sepharose column ~° Procedure. The solutions of avidin and ferritin are mixed and periodate (0.66 ml, 0.1 M solution) is added to a final concentration of 10 raM. The solution is stirred for 30 min on ice, dialyzed against acetatebuffered saline for 6 hr at 4°, and immediately dialyzed overnight at 4° against borate-buffered saline. A fresh solution of sodium borohydride (10 mg/ml in 10 m M NaOH) is prepared, and 0.5 ml is added to the ferritinavidin conjugates in an ice bath. After 1 hr, the solution is dialyzed against PBS. The conjugates are washed twice by centrifugation (100,000 g, 3 hr 6 H. Heitzmann and F. M. Richards, Proc. Natl. Acad. Sci. U.S.A. 71, 3537 (1974). L. Angerer, N. Davidson, W. Murphy, D. Lynch, and G. Attardi, Cell 9, 81 (1976). s E. A. Bayer, E. Skutelsky, D. Wynne, and M. Wilchek, J. Histochem. Cytochern. 24, 933 (1976). 9 STC avidin has been shown by us to contain the full complement of oligosaccharides and is thus similar in its molecular properties to that obtained from Sigma (the former is about 10 times cheaper). Belovo avidin is even cheaper than STC avidin, but its sugar residues are partially degraded. 10 E. A. Bayer, H. Ben-Hur, and M. Wilchek, this volume [8].
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at 4 °) in order to remove free (unconjugated) avidin molecules, and the pellet is resuspended to 1 mg/ml ferritin. The avidin-containing species are separated from the free ferritin by affinity chromatography on an iminobiotin-Sepharose column. 1° The conjugates are passed through a sterile Millipore filter (HA 0.2/zm) and stored in aliquots under sterile conditions at 4 °. Comments. The procedure given here results in a high yield of unitpaired conjugates, which are particularly suitable for precise ultrastructural localization of biotinylated sites associated with subcellular structures. The procedure can, of course, be used to conjugate avidin to other proteins. In cases where the given protein is sensitive to periodate, avidin alone can be preoxidized, the periodate removed by dialysis or gel filtration, and the oxidized avidin reacted further with the desired protein. Owing to the directionality of the above-described chemistry, this procedure is applicable to cases where relatively small conjugates are required.
Avidin- Toxin Conjugates Although we have not included in this volume the use of the avidinbiotin system for therapeutic purposes, the use of avidin-drug or avidintoxin conjugates has recently been seriously considered in studies of cancer therapy. This basic approach is also being used for imaging purposes using conjugates of avidin with radioactive metals. In most of these cases, a biotinylated antibody is first delivered to the target (tissue or organ), and then the avidin-associated probe is introduced. The probe can be either covalently attached to avidin or complexed via a biotin bridge. In some cases, after the biotinylated antibody is interacted with the system, native (underivatized) avidin is delivered, followed sequentially by the biotinylated toxin or radioactive metal. In this chapter, we describe only the preparation of an avidin-toxin conjugate (avidin-ricin A chain). In this case, an active disulfide-containing avidin derivative is interacted with the A chain of ricin (which contains a free sulfhydryl group), thus forming a mixed disulfide conjugate. 11
Reagents Avidin (Belovo or STC Laboratories), nonglycosylated avidin, ~ or streptavidin, 3,1° 10 mg in 4 ml of PBS Ricin A chain (Sigma), 5 mg Acetate-buffered saline (pH 4.5) PBS (pH 7.4) II N. Hashimoto, K. Takatsu, Y. Masuho, K. Kishida, T. Hara, and T. Hamaoka, J. Imrnunol. 132, 129 (1984); K. A. Krolick, J. W. Uhr, and E. S. Vitetta, this series, Vo|. 93 [21].
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N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP, Pharmacia), 10 mg/ml in dimethylformamide Sephacryl S-200 column (1.6 x 88 cm, Pharmacia), equilibrated with PBS Procedure. An aliquot (30 tzl) of SPDP is added to the solution of avidin, and the reaction is allowed to proceed at room temperature for 30 min. The reaction mixture is dialyzed against acetate-buffered saline for 3 hr in order to remove extraneous reagent. Ricin A chain is dissolved in 2.5 ml of acetate-buffered saline, and the solution is dialyzed against the same buffer for 1 hr. The dialyzed solutions of SPDP-modified avidin and ricin A chain are mixed, and the reaction is allowed to proceed overnight at room temperature. The mixture is dialyzed against PBS for 3 hr, and the avidin-ricin A chain conjugates are separated from unreacted avidin and toxin by gel filtration on a Sephacryl S-200 column. The conjugates are passed through a sterile Millipore filter (HA 0.2/~m) and stored in aliquots under sterile conditions at 4 °. Comments. There are many other methods that can be used to couple a toxin to a given carrier. However, in order to obtain an active avidintoxin conjugate, the bond connecting the two should be subject to disassociation. The preferred method is to form a disulfide linkage that would be susceptible to reducing conditions which characterize the intracellular matrix. Internalization is very important for toxin action; the use of cocomplexes containing biotinylated antibody and biotinylated toxin connected via a native (underivatized) avidin bridge may enhance cross-linking on the cell surface and result in internalization of the complexed components into the target cells. The native avidin may also serve as a scavenger to remove rapidly from the circulation free residual toxin and antibody molecules that fail to react with target cells.
Avidin-Penicillinase Conjugates One of the earliest examples of a reagent to be used for conjugating proteins is glutaraldehyde. 12Although this is an example of a homobifunctional reagent and the directionality of the cross-linking cannot be controlled, glutaraldehyde is still a very popular reagent for covalent coupling of proteins. The reason for this (over and above historical precedence and its use out of habit) is simply that the procedure is easy and yields reasonably good results. As an example of this mode of cross-linking, we detail below the coupling of avidin to penicillinase, an interesting enzyme which should find extensive use in immunoassays. One of the possible substrate for12 S. Avrameas,
lmmunochemistry 6, 43 (1969).
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GENERALMETHODOLOGY 400
. . . .
I
Void
. . . .
I
Fer
[18] . . . . Av
200
PCase
300
O
150
200
i00
0 20
I
j
,
-
i00
-
50
,
0
30 Fraction
c~
40
50
number
FIG. 1. Gel filtration of avidin-penicillinase conjugates. Following glutaraldehyde crosslinking of avidin and penicillinase, the reaction mixture was dialyzed and applied to a Sephacryl S-300 column (1.6 x 76 cm) preequilibrated with PBS. The resultant fractions (2 ml) were analyzed for protein content (0) and enzymatic activity (©). Protein content is given as/,~g/fraction, and penicillinase activity represents the reciprocal of time required for the disappearance of color in the substrate solution (hr-l). Void, Fer, Av, and PCase represent the positions of eluted standards for the void volume, ferritin, avidin, and penicillinase, respectively.
mats (presented below) results in the disappearance (as opposed to production) of color, which can be particularly useful in hybridoma technology, pregnancy tests, or any assay where a yes/no answer is required. Reagents
Avidin (Belovo or STC Laboratories), nonglycosylated avidinJ or streptavidin, 3,1° 2 mg ( - 3 0 nmol) dissolved in 0.4 ml PBS Penicillinase from Bacillus cereus (Sigma, -2000 units/mg), 0.5 mg protein ( - 2 0 nmol enzyme) dissolved in 0.1 ml PBS PBS (pH 7.4) Glutaraldehyde solution: 12.4 /zl of an 8% (v/v) solution (Polysciences, Inc., Warrington, PA) is brought to 1.5 ml with PBS Sephacryl S-300 column (1.6 × 76 cm) Substrate solution13:3 ml of 0.5% gelatin (w/v in distilled water), 0.5 13 The substrate solution for penicillinase is prepared immediately before use.
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181
ml of 25 m M iodine in 0.125 M potassium iodide, 0.1 ml of 1% (w/v) soluble starch, and 1 ml of benzylpenicillin [3 mg/ml in 0.1 M phosphate buffer (pH 7)] Procedure. The solutions of avidin and penicillinase are mixed, and the glutaraldehyde solution is added (0.05% final concentration). After 2 hr at room temperature, the slight precipitate that forms is centrifuged, and the supernatant fluids are dialyzed against PBS. The contents of the dialysis tubing are subjected to gel filtration on a Sephacryl S-300 column, and the void-volume fractions are pooled, passed through a sterile Millipore filter (HA 0.2/zm), and stored in aliquots under sterile conditions at 4 °. Comments. Glutaraldehyde-mediated cross-linking of two protein species usually results in the formation of high molecular weight multimers of the two. This is indeed the case with avidin and penicillinase (Fig. 1). Using the conjugation procedure described above, very little free (unconjugated) avidin and penicillinase remain; the largest fraction appears in the void volume (> 1,000,000), and a trail of lower molecular weight multimers can also be recovered. The void-volume fractions (25-27 in Fig. 1) are pooled, and the preparation was found to exhibit high levels of both biotin-binding and penicillinase activities. The preparation is appropriate for immunoassay studies. 14Penicillinase can also be biotinylated and used in conjunction with underivatized avidin to form complexes, similar to those described below. Avidin-Protein Complexes
Avidin and Streptavidin Complexes with Biotinylated Enzymes Preformed complexes between avidin and biotinylated enzymes provide an alternative to classic covalent cross-linking of two proteins to form the corresponding conjugate. The major advantages in using conjugates is that the resultant signal is often enhanced. Moreover, the stock solutions of avidin and many of the biotinylated enzymes can be stored effectively with essentially no deleterious effect on their performance. The preparation of such complexes is convenient, reliable, and reproducible. In forming complexes, a compromise must be reached between the amount of (biotinylated) enzyme which reacts with avidin and the average number of free (available) biotin-binding sites which remain on the avidin molecule. The former is needed to provide a good signal, and the latter is essential for the primary interaction with the desired biotinylated binder 14 R. H. Yolken, this v o l u m e [61].
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(e.g., antibody). The most effective ratio of avidin or streptavidin for complex formation with a given biotinylated enzyme is determined empirically by optimization experiments. In a convenient procedure we designed for this purpose, biotinylated bovine serum albumin is used as a model target system for determining the required conditions for efficient complex formation. Biotinylated alkaline phosphatase (B-AP) is the test enzyme in this example.
Reagents Avidin (Belovo or STC Laboratories), nonglycosylated avidin, l or streptavidin, 3,1° 1 mg/ml in PBS 15 B - A P , 16 500 units/ml in 0.15 M NaC115 PBS (pH 7.4) Bovine serum albumin (BSA), 2% (w/v) in PBS Lysozyme, 2% (w/v) in PBS Biotinylated B S A , 16 1 mg/ml in PBS Substrate solution: 10 mg naphthol AS-MX phosphate (free acid) is dissolved in 200 ~1 of dimethylformamide, and the solution is mixed with a solution containing 30 mg Fast Red dissolved in 100 ml of 0.1 M Tris-HCl (pH 8.4) Optimization Procedure. Complexes consisting of different ratios of avidin (or streptavidin) and B - A P are prepared in the following manner. The stock solution of avidin is diluted to 1, 3, 10, and 30/xg/ml (4 ml total solution each).~7 Similarly, the stock solution of B - A P is diluted (e.g., 1, 2, 5, and 10 units/ml, 4 ml total). ~8A 1-ml sample of each concentration of avidin is combined with an equivalent volume of one of the B - A P solutions in matrix fashion, yielding 16 separate solutions of varying ratios and quantities of avidin to B-AP. The solutions are allowed to stand for about 30 min at room temperature before being incubated with dot blots containing the target material. The biotinylated target protein is applied to dot blots in the following manner. Samples (1 /xl) containing serial dilutions of biotinylated BSA, are applied onto 16 dot blots. 19 The strips are rinsed with PBS, treated with quenching solution, z° and rinsed again, and each blot is incubated in one of the above-described preformed complex preparations. After 30 15 Stock solutions o f avidin (streptavidin) and B - A P are stored in aliquots at - 2 0 °. The stock solutions are u s e d no m o r e t h a n twice (thawed, refrozen, and rethawed). 16 E. A. B a y e r and M. Wilchek, this v o l u m e [14]. 17 L y s o z y m e solution is u s e d as a diluent for avidin, and B S A is u s e d for streptavidin. ~8 B S A is u s e d as a diluent for B - A P . ~9 E. A. Bayer, H. B e n - H u r , and M. Wilchek, this volume [48]. 2o B S A is u s e d as q u e n c h e r for streptavidin-based complexes, and a 1 : 1 mixture of B S A and l y s o z y m e is u s e d for avidin-based complexes.
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183
B-AP (Ulml) 2
I
5
I0
I J
i i
•
i~l
f
hill
o •
0 l, e
i
0 0
Q •
0
0
0 (j
0 oe
III
qllJ e
tqDO
I
Q' • e
0
q~ i '
30 t
FIG. 2. Dot-blot optimization of complex formation between avidin and B-AP. Onto each nitrocellulose strip, 1-/~1 s a m p l e s containing serial dilutions (top row: 1000, 300, 100, 30, 10, and 3 ng//xl; b o t t o m row: 1, 0.3, 0.1, etc.) of biotinylated B S A were applied. The strips were rinsed, q u e n c h e d , rinsed again, and incubated in solutions containing the different c o m p l e x e s . T h e designated a m o u n t s indicate the initial concentrations of either B - A P or avidin solutions that were c o m b i n e d 1 : 1 and applied to the corresponding nitrocellulose strip. Following incubation, the strips were rinsed, and substrate solution was added.
min, the strips are rinsed again with PBS, and substrate solution is added. Bands usually appear within 30 min; at the desired amplification, the reaction is terminated by washing the strips with tap water. Standard Procedure. Once the system has been optimized for a given biotinylated enzyme preparation, the procedure for complex formation can be standardized using a given ratio of components. Using the B - A P prepared in our laboratory, ~6we found the following protocol to result in optimal levels of complexation of the enzyme with avidin or streptavidin. A 100-fold dilution 17,18 of the above stock solutions of both avidin (or streptavidin) and B - A P are combined and used after a 30-min incubation at room temperature. Comments. The optimal ratio for complex formation corresponds to about 2/xg avidin per unit of B - A P (Fig. 2); initial concentrations (before mixing) of 10/~g/ml avidin and 5 units/ml B - A P are generally used. At relatively low avidin-to-B-AP ratios (i.e., 1 /xg/ml and 10 units/ml, respectively), little labeling can be observed, presumably since most of the biotin-binding sites of avidin are involved in complex formation and few free sites are available to combine with the biotinylated target protein. Conversely, at high avidin-to-B-AP ratios (i.e., 30/zg/ml and 1 unit/ml, respectively), the labeling pattern is also impaired, apparently because
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extraneous uncomplexed avidin molecules compete with the complexes, thereby reducing the amount of enzyme available for detection. It should be noted that the concentration of complexes introduced into the system should be regulated in order to reduce the level of nonspecific binding. Specifically, high background levels are observed at B-AP concentrations higher than 10 units/ml, and such conditions should be avoided. The optimal ratio for streptavidin-containing complexes is identical to that of avidin. The use of streptavidin usually results in much lower levels of either nonspecific binding or background, and it is generally recommended when available. For new batches of biotinyl enzyme, such optimization experiments should always be performed. Stored samples of reagents should also be subjected periodically to such experiments; the complexes themselves are not stable for long periods of time and cannot be stored.
Avidin and Streptauidin Hydrazides Hydrazide derivatives of avidin and streptavidin have been shown to be effective for selectively labeling glycoconjugates on blots. 19,2~The avidin hydrazide is combined with a biotinylated enzyme at an optimized ratio to form an amplified enzyme-hydrazide preparation, which can be used to label periodate-oxidized or galactose oxidase-treated saccharides.
Reagents Avidin (Belovo or STC Laboratories), nonglycosylated avidin, 1 or streptavidin, 3,1° 50 mg/ml Adipic acid dihydrazide (Sigma), 160 mg Water-soluble carbodiimide, 22 160 mg Distilled water 5 N HC1 PBS (pH 7.4) Procedure. The dihydrazide is dissolved by heating in 5 ml of distilled water, and the pH is adjusted to 5 with HC1. The solution is added to solid avidin or streptavidin (50 mg of the desired protein). The water-soluble carbodiimide is added in solid form, and the reaction is allowed to proceed for 3 hr at room temperature with periodic 30-min adjustments of the pH to 5. The avidin hydrazide is dialyzed exhaustively against PBS. The product is diluted to 50 ml with PBS, passed through a sterile Millipore filter (HA 0.2/xm), and stored in 1-ml aliquots under sterile conditions at 4°. Both avidin and streptavidin hydrazide are stable for years under these conditions. 21 E. A. Bayer, H. Ben-Hur, and M. Wilchek, Anal. Biochem. 161, 123 (1987). 22 l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Sigma).
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185
Complexes of Biotinylated Enzymes with Avidin (or Streptavidin) Hydrazides. To a solution of avidin hydrazide or streptavidin hydrazide (15 /xg/ml, dissolved in 2% w/v lysozyme in PBS) is mixed an equal volume of biotinyl alkaline phosphatase 15,~6(7.5/xg/ml). 23 Comments. The optimal ratio of hydrazide probe to biotinyl enzyme for complex formation is determined by dot-blot optimization experiments similar to that described above for avidin-enzyme complexes, except that the target molecule consists of a suitable glycoconjugate (e.g., fetuin) which is oxidized by enzymatic or chemical means.19 The ratio of reagents is based on the results of such an experiment. As mentioned above, for new batches of biotinyl enzyme and avidin (or streptavidin) hydrazide, such optimization experiments should always be performed. Stored samples of reagents should also be subjected periodically to such experiments. The solution is applied to blots about 30 min after initiation of complex formation; the complexes themselves are not stable for long periods of time and cannot be stored. Derivatization of a protein via its carboxyl group results in increased pI values; a neutral protein such as streptavidin becomes basic, and a basic protein such as avidin becomes even more basic. This phenomenon generates high levels of nonspecific binding which can easily be countered by quenching with appropriate reagents. 23 Thus, in lieu of using albumin (a negatively charged protein) as a quencher, we employ lysozyme (the pI of which is even higher than that of avidin), zl
Immobilized Avidins Avidin can be immobilized to solid matrices in many different ways. The classic method involving CNBr activation of Sepharose has been described explicitly in the past. 24 In this section, we present two alternative methods for immobilization of avidin to Sepharose.
Cyano-Transfer Activation of Sepharose In the first method, the use of the extremely hazardous CNBr for activation is precluded by using a cyano-transfer derivative or intermediate. 25 23 For complexes containing avidin hydrazide, 2% lysozyme is used as a diluent; for complexes containing streptavidin hydrazide, the diluent consists of 2% BSA. 24 E. A. Bayer, E. Skutelsky, and M. Wilchek, this series, Vol. 62 [55]; E. A. Bayer and M. Wilchek, Methods Biochem. Anal. 26, 1 (1980); A. Bodanszky and M. Bodanszky, Experientia 26, 237 (1970). 25 M. Wilchek, T. Miron, and J. Kohn, this series, Vol. 104 [I].
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GENERAL METHODOLOGY
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Reagents Avidin (Belovo or STC Laboratories), nonglycosylated avidin, l or streptavidin, 3,~° 200 mg in 100 ml of 0.1 M sodium bicarbonate Sepharose 4B or CL-4B, 50 ml of swollen gel N - C y a n o t r i e t h y l a m m o n i u m tetrafluoroborate (CTEA), 540 mg, or 1cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP), 225 mg z6 (Sigma) 0.2 M Triethylamine (aqueous solution) Acetone Distilled water Washing medium: acetone : 0.I N HCI (1:1, v/v) Storage medium: acetone : dioxane : water (60 : 35 : 5, v/v) Coupling medium: 0.1 M sodium bicarbonate (pH 8.5) PBS (plus 0.1% sodium azide, p H 7.4) Procedure. The Sepharose is washed with water, 30% acetone, and 60% acetone, successively, and resuspended in 10 ml of 60% acetone. The suspension is cooled to 0 °, and the desired cyano-transfer reagent is added. The triethylamine solution (5.4 ml for the CTEA-activated resin or 1.8 ml for the CDAP-activated resin) is then added dropwise with vigorous stirring. After 2 min, the reaction mixture is transferred to 100 ml of ice-cold washing medium. The resin can be maintained in this manner for about 1 hour without loss of active groups. 27 The activated resin is washed with large volumes of cold water, followed by a rapid wash with coupling medium. The solution of avidin is mixed with the resin, and the interaction is carried out for 1 hr at room temperature and overnight at 4 ° . The resin is washed with coupling buffer and PBS, 28 and stored in PBS. Comments. The above procedure results in about 80-90% coupling, z8 yielding about 3.5 mg avidin/ml swollen gel. The end products of the reaction are identical to those of the C N B r activation procedure, and a certain amount of leakage of avidin may thus be expected. 25 Nevertheless, these reagents are nonvolatile solid cyanoderivatives which can be stored and handled without use of a fume hood; they should therefore be used in place o f C N B r since they are more convenient, more efficient and, of course, safer. 26Both reagents can be obtained from Sigma. 27For prolonged storage of cyano-transfer-activated Sepharose, the resin is washed extensively with storage medium and maintained at -20 °. When required, the activated resin is reswollen for 5 min in cold washing medium and washed according to the procedure for immediate coupling. 2s In the case of CTEA-activated resins, the A2s0of the washes can be followed in order to determine the extent of coupling.
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AVIDIN-CONTAININGPROBES
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p-Nitrophenyl Chloroformate-Activated Sepharose One of the major problems with CNBr (or cyano-transfer) activation of Sepharose is the observed leakage of ligand owing to the instability of some of the bonds formed. Alternative procedures have therefore been developed which provide improved chemical attachment of proteins and other ligands. 25 One of the most effective is the use of chloroformates that result in activated carbonate groups on the resin. Subsequent interaction with amines yields stable and uncharged carbamate (urethane) derivatives.
Reagents Avidin (Belovo or STC Laboratories), nonglycosylated avidin, l or streptavidin, 3,~° 200 mg in 100 ml of 0.1 M sodium bicarbonate Sepharose CL-4B (Pharmacia), 50 ml p-Nitrophenyl chloroformate (Aldrich Chemical Company, Milwaukee, WI), 3 g 4-Dimethylaminopyridine (Sigma), 2.1 g Acetone (absolute) 2-Propanol Distilled water Coupling medium: 0. I M sodium bicarbonate (pH 8.5) PBS (plus 0.1% sodium azide, pH 7.4) Procedure. The activation procedure is carried out as described elsewhere in this volume, l° The solution of avidin is then mixed with the washed, activated resin; the interaction is carried out for 1 hr at room temperature and overnight at 4 °. The resin is washed successively with water, 0.2 M acetic acid, water, and (rapidly) with 10 mM NaOH. The resin is then washed exhaustively with distilled water, followed by coupling buffer and PBS. The immobilized avidin is stored in PBS. Comments. Using the above procedure, about 3 mg avidin can be coupled per milliliter of Sepharose. The covalent bonds formed are very stable, and leakage is negligible. In other respects, the performance of such columns is equivalent to that of CNBr-activated resins.
