ANALYTICAL
BIOCHEMISTRY
186,69-73
(1990)
Copper Iodide Staining and Determination Adsorbed to Microtiter Plates’
of Proteins
Douglas D. Root and Emil Reisler Molecular Biology Institute and Department of Chemistry University of California, Los Angeles, California 90024
Received
September
and Biochemistry,
29,1989
Copper iodide staining and determination of proteins adsorbed to polystyrene microtiter plates are described. The minimum amount of copper iodide-stained protein detected in densitometric measurements is approximately 20 pg/mm’. Enzyme immunoassay readers may also be used for the determination of copper iodidestained proteins, but are less sensitive than densitometers. The densitometric readings of copper iodidestained proteins vary linearly with the amount of protein present as verified by enzymatic and radioactive probes. Staining is complete in 2-3 min and may be removed by a 30-min treatment with EDTA without loss of adsorbed protein or immunoreactivity. The exact amount of protein adsorbed to microtiter plate wells can be measured by using protein bound and stained on nitrocellulose as a calibration curve. Copper iodide staining is a rapid, convenient, and inexpensive alternative to radioactive measurements of similar parameters. Q 1990 Academic Press, Inc.
Copper iodide staining of proteins has recently been introduced as a highly sensitive protein stain for Western blots on nitrocellulose and nylon membranes (1). The stain utilizes the copper binding properties of proteins under alkaline conditions (2) and a precipitation reaction of reddish-brown copper iodide (1). Its reversibility, low cost, speed, and simplicity make it attractive for use in other assays as well. Protein staining for quantitative and qualitative evaluation of antigens bound to microtiter plates has been addressed for another solid
‘This work was supported by the National Institutes of Health Grant AR 22031 and NSF Grant DMB 89-05363. Douglas D. Root was supported by a USPHS National Institutional Research Service Award CA-09056. 0003.2697/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
Inc. reserved.
phase protein stain (3). The use of copper iodide for this purpose was therefore suggested (1). Another ultrasensitive protein stain, silver-enhanced colloidal gold, has been used to evaluate the consistency of protein binding to microtiter plates (3). Inconsistent antigen adsorption can be responsible for inaccurate enzyme immunoassay results (3). Thus, protein staining could be a useful control for serological laboratory tests. Copper iodide, like silver-enhanced colloidal gold, may be used to visualize proteins adsorbed to microtiter plates. However, copper iodide is quicker, simpler, and less expensive than silver-enhanced colloidal gold (1,3). In addition, quantitations by silver-enhanced colloidal gold run the risk of sensitivity loss before the microtiter plate well is saturated with protein (3). Several studies have indicated that proteins vary in their adsorption properties (4-7). Cantarero et al. have defined a “range of independence” as the range of concentrations over which the amount of protein bound to polystyrene varies linearly with the amount of protein applied to the well (4). Above these concentrations, adsorption is inversely proportional to the protein concentration and is believed to be mediated by protein-protein interactions (4). Knowledge of this range of independence is necessary to avoid wasting precious antigen when coating the well and to allow all proteins in a mixture to be represented in the adsorbed material (4). A quantitative protein stain can be used to determine the range of independence for any protein of interest. Binding studies by enzyme immunoassay procedures could be both confirmed and enhanced by a staining method which can determine the exact amount of protein in a microtiter plate well. Three areas where such a method might be utilized include (i) the study of lateral interactions between antibodies as they bind to different concentrations of antigen immobilized on solid phases (&lo), (ii) the evaluation of theories of diffusion-limited reactions of proteins binding to microtiter plate wells (lO,ll), and (iii) the determination of both relative and 69
70
ROOT
AND
absolute binding constants by enzyme immunoassay procedures (3). This study evaluates the efficacy of copper iodide staining of proteins adsorbed to polystyrene microtiter plates. A method for determination of the amount of adsorbed protein is also presented. MATERIALS
AND
METHODS
Materials Flat-bottom polystyrene Immulon 1 and 2 microtiter plates were purchased from Dynatech Laboratories, Inc. (Chantilly, VA). Nitrocellulose membranes (BA85) were procured from Schleicher & Schuell (Keene, NH). Bovine albumin (BSA)2 was obtained from the United States Biochemical Corp. (Cleveland, OH). Rabbit muscle actin was prepared by the method of Spudich and Watt (12). Site-specific polyclonal antibodies against actin were a gift from Dr. Gargi DasGupta and have been described previously (13). Alkaline phosphatase-conjugated goat anti-rabbit IgG was bought from Sigma Chemical Co. (St. Louis, MO). ScintiVerse Bio-HP (SO-X-14) was from Fisher (Fairlawn, NJ). [14C]Formaldehyde (55 mCi/mmol) was procured from NEN Research Products, DuPont (Boston, MA). Bicinchoninit acid protein assay (BCA) was from Pierce Chemical Co. (Rockford, IL). Coomassie brilliant blue R-250 was obtained from Schwarz/Mann Biotech (Cleveland, OH). Naphthol blue black and the Bradford protein assay reagent were purchased from Bio-Rad (Richmond, CA). Household 3-in-one lubricating oil was from Boyle-Midway, Inc. (New York, NY). Copper sulfate was Baker Analyzed, T. J. Baker, Inc. (Phillipsburg, NJ). All other reagents were of analytical grade. Protein Adsorption and Dot Blotting Proteins were diluted to appropriate concentrations in phosphate-buffered saline, pH 7.6 (PBS), and applied in 200 ~1 aliquots to 96-well Immulon 1 or 2 microtiter plates. Copper iodide staining determined that 4 h at 22°C gave maximal protein adsorption to plates (data not shown). Plates were then washed five times by submersion in deionized water and allowed to dry. Dot blotting was performed on nitrocellulose membranes which were soaked in PBS for 5 min and then allowed to dry. Proteins diluted in PBS were applied in 5-~1 drops and allowed to dry before staining.
REISLER
potassium iodide (20 g), sodium potassium tartrate (36 g), and 80 ml of deionized water were mixed into a fine slurry. Sodium hydroxide (10 g) was then dissolved into the solution which was subsequently allowed to stand at room temperature for 30 min. After a reddish precipitate settled, 70 ml of the supernatant was discarded. The remaining solvent and precipitate were mixed together prior to use. An excess of reagent was added to each microtiter plate well for an appropriate incubation time. The plate was then washed five times in deionized water and allowed to dry. Dot blots on nitrocellulose membranes were stained as previously described (1) except that 2-min incubations were used. Quantitation was performed with a Biomed Instruments (Fullerton, CA) Model SL-504-XL soft laser scanning densitometer equipped with an integrator and interfaced to an Apple IIe computer. Alternatively, a Dynatech (Chantilly, VA) MR600 microplate reader was used at a wavelength of 650 nm. The copper iodide stain was removed by filling the wells with 4 mM EDTA in PBS for 30 min. Plates were then washed with deionized water and allowed to dry. Coomassie and amido black staining reagents were prepared as described before (14). BCA protein assays were performed according to the manufacturer’s instructions and as originally described by Smith et al. (2). Enzyme immunoassays were performed by standard methods (15) but using 200-~1 aliquots per well. Radiolabeling and Counting BSA was labeled with 14Cby reductive methylation with [14C]formaldehyde (16). A 1 mg/ml BSA solution in 20 mM sodium cyanoborohydride and 100 mM Hepes, pH 9.0, was reacted for 24 h at 22°C with 6 mM [‘“Clformaldehyde (55 mCi/mmol). Three volumes of 10% trichloroacetic acid was added, and the precipitated protein was isolated by centrifugation. The pellet was washed once and redissolved in PBS. The protein concentration was determined by the BCA protein assay. The specific activity was determined to be 3.6 Ci/mmol by counting in 3 ml of ScintiVerse. [14C]BSA adsorbed to microtiter plates was quantified by excising the well with a hot knife (Cooper Tools, Apex, NC), dissolving the polystyrene overnight in ScintiVerse, and liquid scintillation counting (Beckman, LS 8100, Irvine, CA). Radiolabeled and native BSA had similar saturation plots on microtiter plates as judged by copper iodide staining (data not shown).
Staining and Enzyme Immunoassays The copper iodide staining reagent was prepared as described before (1). Copper sulfate pentahydrate (12 g), ’ Abbreviations used: BSA, bovine serum sity; PBS, phosphate-buffered saline; BCA, assay; IgG, immunoglobulin G.
albumin; OD, optical denbicinchoninic acid protein
Calibration of Microtiter
Plates
[14C]BSA was adsorbed to microtiter plate wells at desired concentrations and stained with copper iodide. [14C]BSA, between lo-100 ng/5 ~1 drop, was dot blotted onto a nitrocellulose membrane and stained with copper iodide. The dot blots were excised using a standard hole
COPPER
2.0
110
Incubation
3.0
IODIDE
4.0
STAINING
5.0
Time (min)
FIG. 1. Time course of copper iodide staining of microtiter plates. BSA (2 rg) was adsorbed to the microtiter plate, stained with copper iodide for the indicated periods of time, washed, and read with a densitometer. Bars represent the standard deviations (n = 3-4 wells).
puncher with a 6-mm diameter and placed into empty microtiter plate wells. Blank areas on the nitrocellulose membrane were punched out and used to cover wells with polystyrene-bound BSA that had been stained with copper iodide. Blank wells were also covered with blank nitrocellulose to measure the background. Five microliters of household 3-in-one lubricating oil was added to each well to render the nitrocellulose transparent (17). Staining was immediately quantified by densitometry and compared to results obtained by radioactivity measurements. RESULTS
AND
OF
MICROTITER
71
PLATES
a mixture of proteins within their range of independence allows all the proteins in the mixture to be represented on the plate in a consistent, although not necessarily stoichiometric, manner (4). A profile of the saturation curve (Fig. 2) enables the selection of optimal adsorption conditions and thus avoids considerable waste of the applied protein. To determine this profile accurately, the protein stain used must give a linear increase of density with the amount of protein present. To demonstrate the quantitative nature of copper iodide staining, the amount of absorbed protein was measured enzymatically and with [ ‘“C]BSA. Figure 3 shows good agreement between the alkaline phosphatase reaction and the copper iodide staining. The correlation coefficient was r2 = 0.982. Radioactivity measurements agreed even more closely with the densitometry of copper iodide staining as shown in Fig. 4. The correlation coefficient was r2 = 0.994. Thus, copper iodide staining is effective for quantitative measurements, and its density varies linearly with the amount of protein present. Since copper iodide staining is quantitative, the density (rig/mm’) of copper iodide-stained protein on a microtiter plate can be determined by the following procedure: (i) Dot blot the protein of interest in known amounts to form a standard curve. Typically lo-100 ng/ 5 ~1 drop, at least in quadruplicate, is dot blotted onto nitrocellulose and stained with copper iodide. (ii) Determine the average diameter of the dots and the average area covered by protein, typically 15.9 -t 1.1 mm2 (n = 43). (iii) Excise the dots with a hole puncher and place them in blank wells in the microtiter plate. To equalize
DISCUSSION
One of the attributes of copper iodide staining is its speed. Figure 1 shows a time course for copper iodide staining of BSA on microtiter plates. Maximal intensity of the stain was reached in 2-3 min; therefore, this incubation time was used for subsequent experiments. Incubations for up to 15 min did not result in any significant losses of staining intensity when compared to the plateau levels reached after 3 min of staining (data not shown). In principle, as shown in Fig. 2, densitometric and microtiter plate reader measurements of copper iodide staining can give similar results. The densitometric analysis, however, is more sensitive and yields more consistent results. Two possible reasons for the greater sensitivity of the densitometer are (i) a greater area of the microtiter plate well can be scanned and integrated, and (ii) the unfiltered densitometer integrates the entire visible adsorption spectrum of copper iodide, while the microtiter plate reader measures only a single wavelength. Figure 2 also demonstrates the range of independence of BSA adsorption. The value of 200 ng/200 ~1 found here is similar to the value of 1000 ng/lOOO ~1 found by Cantarero et al. (4). These authors found that adsorbing
120
-I
0
71
100
200
BSA
300
Applied
400
500
(ng)
FIG. 2. Comparison of densitometric and the microtiter plate reader measurements of copper iodide-stained protein. BSA was coated on to the microtiter plate wells at the indicated amounts per 200 ~1, stained with copper iodide, and read with both a densitometer and a microtiter plate reader. Open circles represent densitometer readings, while closed triangles are values from the microtiter plate reader (the maximum OD was 0.057 on the microtiter plate reader). Bars represent the standard deviations (n = 3 wells). Standard deviations for closed triangles are to the right of the symbol for clarity. Closed triangles have also been shifted to the right when they are at points identical with the circles.
72
ROOT
0
40
20 IgG
60 Applied
SO
AND
100
(ng)
the background, place blank nitrocellulose over both empty control wells and sample wells where the protein adsorbed directly to the microtiter plate has been stained with copper iodide. (iv) Add 5 ~1 of household 3in-one oil to render the nitrocellulose transparent and read with a densitometer.
60
o500
1000 BSA
Applied
0.0
2.0
4.0 BSA
FIG. 3. Comparison of copper iodide staining with enzymatic assays. Alkaline phosphatase-conjugated goat anti-rabbit IgG was adsorbed to the microtiter plates at the indicated amounts per 200 ~1, developed, and read like an enzyme immunoassay. The plate was then washed, stained with copper iodide, and read. Open circles represent copper iodide staining, while closed triangles are from enzymatic readings (the maximum OD was 1.368 on the microtiter plate reader). Bars represent standard deviations (n = 5 wells). Standard deviations for closed triangles are to the right of the symbol for clarity. Closed triangles have also been shifted to the right when they are at points identical with the circles.
0
REISLER
1500
2000
(ng)
FIG. 4. Comparison of copper iodide staining with radioactive assays. [%]BSA was adsorbed to microtiter plates at the amounts indicated per 200 ~1. The plate was stained with copper iodide and read. Then the wells were excised, and the amount of protein was determined by liquid scintillation counting. The lowest amount of protein detected by copper iodide staining was 3 ng per well. Open circles represent copper iodide staining, and closed triangles represent radioactive measurements (the maximum radioactivity was 280 Bq/well from [%]BSA with asp act of 3.6 Ci/mmol). Bars show the standard deviations (n = 5 wells). Standard deviations for closed triangles are to the right of the symbol for clarity. Closed triangles have also been shifted to the right when they are at points identical with the circles.
6.0
(rig/mm*)
FIG. 5. Calibration of microtiter plates by copper iodide staining. [‘%]BSA was adsorbed to microtiter plate wells (0,100,200,300,500, and 2000 ng/200 PI), washed, stained with copper iodide, and covered with blank nitrocellulose. Dot blots of [‘%]BSA (10,20,40,60,80, and 100 ng) were stained with copper iodide and placed in blank microtiter plate wells. Five microliters of household 3-in-one oil was added, and the plates were read by densitometry. The amount of protein coating the microtiter plate wells was verified by liquid scintillation counting. Densities were normalized to the area covered by protein. Open circles represent microtiter plate-bound protein (the maximum radioactivity of the open circles was 105 Bq/well from [14C]BSA with a sp act of 560 mCi/mmol), while closed circles represent nitrocellulose-bound protein. Bars show the standard deviations (n = 5-6).
From the density (rig/mm’) of protein on the microtiter plate, the total amount of adsorbed protein can then be calculated based on the dimensions of the well and the volume of protein solution originally added to it. For a 6.4-mm diameter flat-bottom well loaded with 200 ~1 of protein solution, the area covered by protein is 3.14r2 + (2V/r) = (3.14)(0.32 cm)’ + (2)(0.2 cm3)/0.32 cm = 1.57 cm’. Where r is the radius of the flat-bottom well, and V is the volume of the protein solution added for adsorption. This area can then be multiplied by the amount of protein per area determined from the standard curve of copper iodide-stained protein on nitrocellulose to render the total protein adsorbed to the microtiter plate. This method was used in Fig. 5 to determine the amounts of BSA adsorbed to a microtiter plate, and radioactivity measurements were used to verify the results. The accuracy of this method is based on two observations: (i) All of the protein applied to nitrocellulose by dot blots will be tightly bound, when saturation is not approached (M-21). (ii) Copper iodide staining did not cause a significant loss of protein from microtiter plates or nitrocellulose membranes as measured by radioactivity (data not shown). Correlation coefficients for radioactivity versus densitometric measurements of copper iodide-stained protein on nitrocellulose membranes or microtiter plates were r2 = 0.997 and r2 = 0.988, respectively. Thus, this copper iodide staining method of protein determination can substitute for radioactive measurements of exact amounts of bound protein.
COPPER
IODIDE
STAINING
The sensitivity of copper iodide staining on microtiter plates can be calculated from Fig. 4 as 20 pg/mm2. Silverenhanced colloidal gold may be more sensitive in detecting protein on microtiter plates (3), but its quantitative characteristics are more complex. In the case of horseradish peroxidase-conjugated IgG adsorbed to microtiter plates, the maximal intensity of silver-enhanced colloidal gold was reached at concentrations below enzymatitally detectable IgG (3). Copper iodide staining is, however, readily quantifiable in the same range of detection as enzyme assays (Fig. 3). Other attempts to detect and quantify proteins adsorbed to microtiter plates have had limited success. The BCA assay, like copper iodide, is copper-based (2), but it works only in solution and is not permanent (22). The BCA assay is less sensitive than copper iodide (22). In addition, it has not yet been shown that its extinction coefficients are the same for bound proteins as for proteins in solution (22). Ellipsometry has been used to detect adsorbed proteins (9,lO) but is also less sensitive than copper iodide staining and cannot be applied directly to microtiter plates. The Bradford protein assay, amido black, and Coomassie brilliant blue stains were unable to detect proteins adsorbed to microtiter plates (data not shown). Thus, copper iodide staining appears to be the most sensitive and proven method for estimating protein amounts on microtiter plates without the use of radioactivity. Copper iodide staining can be advantageous in comparative binding studies between different proteins. Take, as an example, an antibody being tested for its relative binding affinity to two different antigens. Microtiter plates could be coated with either of the two antigens, and the exact amount of adsorbed antigen could be assessed by copper iodide staining. A direct binding assay could then be normalized by the amount of antigen present. Such an experimental design would avoid problems of crosslinking by the antibody to both types of antigens which might occur in competitive binding assays. The reversibility of copper iodide staining of protein is another virtue of this procedure (1). A 30-min incubation with 4 mM EDTA in PBS is adequate to remove the stain from proteins on microtiter plates (data not shown). After actin was stained and destained with copper iodide, it was recognized by and would bind site-specific polyclonal antibody with an intensity similar to that of unstained actin (data not shown). Therefore, evaluation of the adsorbed antigen can be made both visually and quantitatively prior to reaction with the antibody. Copper iodide staining has many important attributes which make it valuable for use on microtiter plates. It
OF
MICROTITER
73
PLATES
is highly sensitive, quantitative, reversible, inexpensive, and convenient. In addition, the calibration technique presented here enables the accurate and sensitive determination of the amount of protein coating a microtiter plate. This procedure has many possible research applications particularly in the theory and practice of binding studies (6-11). Copper iodide staining may even be valuable for routine serological assays and quality control by visualizing the antigen coating at a lower cost than other techniques (3). ACKNOWLEDGMENT We thank to actin.
Dr. Gargi
DasGupta
for providing
site-specific
antibodies
REFERENCES 1. Root, D. D., and Reisler, E. (1989) Anal. &o&em. 181.250-253. 2. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gart ner, F. H., Provenzano, M. D., Fujimoto, N. M., Goeke, B. J., Olson, B. J., and Kenk, D. C. (1985) Anal. Biochem. 150,76-85. 3. Gibbs, J., and Root, D. M. (1988) Costar Research Newsletter, Vol. 1, Issue 2. 4. Cantarero, L. A., Butler, J. E., and Osborne, J. W. (1980) Anal. Biochem. 105,375-382. 5. Vogt, R. F., Phillips, D. L., Henderson, L. O., Whitfield, W., and Spierto, F. W. (1987) J. Immunol. Methods 101,43-50. 6. Fair, B. D., and Jamieson, A. M. (1980) J. Colloid Interface Sci. 77,525-534. I. Lee, R. G., Adamson, C., and Kim, S. W. (1974) Thromb. Res. 4, 485-490. 8. Nygren, H. (1988) J. Zmmunol. Methods 114,107-114. 9. Werthen, M., and Nygren, H. (1988) J. Immunol. Methods 115, ‘7-78. 10. Nygren, H., Kaartinen, M., and Stenberg, M. (1986) J. Zmmunol. Methods 92,219-225. 11. Stenberg, M., Werthen, M., Theander, S., and Nygren, H. (1988) J. Zmmunol. Methods 112,23-29. 12. Spudich, K., and Watt, S. (1971) J. Biol. Chem. 246,4866-4871. 13. Miller, L., Kalnoski, M., Yanossi, Z., Bulinski, J. C., and Reisler, E. (1987) Biochemistry 26,6064-6070. 14. Robyt, J. F., and White, B. J. (1987) Biochemical Techniques: Theory and Practice, pp. 144-145, Brooks/Cole Publishing Co., Monterey, CA. 15. Atherton, B. T., and Hynes, R. 0. (1981) Cell 25,133-141. 16. Jentoft, 4365. 17. Maciewicz,
N., and Dearborn,
D. G. (1979)
R. A., and Knight,
J. Biol
P. J. (1988)
Chem.
Anal.
254,4359-
Biochem.
175,
85-90. 18. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76.4350-4354. 19. Lin, W., and Kasamatsu, H. (1983) Anal. Biochem. 128,302-311. 20. Salinovich, O., and Montelaro, R. C. (1986) Anal. Biochem. 156, 341-347. 21. Enosawa, S., and Ohashi, A. (1987) Anal. Biochem. 160,211-216. 22. Sorensen, K., and Brodbeck, U. (1986) J. Immunol. Methods 95, 291-293.
BIOCHEMISTRY
186,69-73
(1990)
Copper Iodide Staining and Determination Adsorbed to Microtiter Plates’
of Proteins
Douglas D. Root and Emil Reisler Molecular Biology Institute and Department of Chemistry University of California, Los Angeles, California 90024
Received
September
and Biochemistry,
29,1989
Copper iodide staining and determination of proteins adsorbed to polystyrene microtiter plates are described. The minimum amount of copper iodide-stained protein detected in densitometric measurements is approximately 20 pg/mm’. Enzyme immunoassay readers may also be used for the determination of copper iodidestained proteins, but are less sensitive than densitometers. The densitometric readings of copper iodidestained proteins vary linearly with the amount of protein present as verified by enzymatic and radioactive probes. Staining is complete in 2-3 min and may be removed by a 30-min treatment with EDTA without loss of adsorbed protein or immunoreactivity. The exact amount of protein adsorbed to microtiter plate wells can be measured by using protein bound and stained on nitrocellulose as a calibration curve. Copper iodide staining is a rapid, convenient, and inexpensive alternative to radioactive measurements of similar parameters. Q 1990 Academic Press, Inc.
Copper iodide staining of proteins has recently been introduced as a highly sensitive protein stain for Western blots on nitrocellulose and nylon membranes (1). The stain utilizes the copper binding properties of proteins under alkaline conditions (2) and a precipitation reaction of reddish-brown copper iodide (1). Its reversibility, low cost, speed, and simplicity make it attractive for use in other assays as well. Protein staining for quantitative and qualitative evaluation of antigens bound to microtiter plates has been addressed for another solid
‘This work was supported by the National Institutes of Health Grant AR 22031 and NSF Grant DMB 89-05363. Douglas D. Root was supported by a USPHS National Institutional Research Service Award CA-09056. 0003.2697/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
Inc. reserved.
phase protein stain (3). The use of copper iodide for this purpose was therefore suggested (1). Another ultrasensitive protein stain, silver-enhanced colloidal gold, has been used to evaluate the consistency of protein binding to microtiter plates (3). Inconsistent antigen adsorption can be responsible for inaccurate enzyme immunoassay results (3). Thus, protein staining could be a useful control for serological laboratory tests. Copper iodide, like silver-enhanced colloidal gold, may be used to visualize proteins adsorbed to microtiter plates. However, copper iodide is quicker, simpler, and less expensive than silver-enhanced colloidal gold (1,3). In addition, quantitations by silver-enhanced colloidal gold run the risk of sensitivity loss before the microtiter plate well is saturated with protein (3). Several studies have indicated that proteins vary in their adsorption properties (4-7). Cantarero et al. have defined a “range of independence” as the range of concentrations over which the amount of protein bound to polystyrene varies linearly with the amount of protein applied to the well (4). Above these concentrations, adsorption is inversely proportional to the protein concentration and is believed to be mediated by protein-protein interactions (4). Knowledge of this range of independence is necessary to avoid wasting precious antigen when coating the well and to allow all proteins in a mixture to be represented in the adsorbed material (4). A quantitative protein stain can be used to determine the range of independence for any protein of interest. Binding studies by enzyme immunoassay procedures could be both confirmed and enhanced by a staining method which can determine the exact amount of protein in a microtiter plate well. Three areas where such a method might be utilized include (i) the study of lateral interactions between antibodies as they bind to different concentrations of antigen immobilized on solid phases (&lo), (ii) the evaluation of theories of diffusion-limited reactions of proteins binding to microtiter plate wells (lO,ll), and (iii) the determination of both relative and 69
70
ROOT
AND
absolute binding constants by enzyme immunoassay procedures (3). This study evaluates the efficacy of copper iodide staining of proteins adsorbed to polystyrene microtiter plates. A method for determination of the amount of adsorbed protein is also presented. MATERIALS
AND
METHODS
Materials Flat-bottom polystyrene Immulon 1 and 2 microtiter plates were purchased from Dynatech Laboratories, Inc. (Chantilly, VA). Nitrocellulose membranes (BA85) were procured from Schleicher & Schuell (Keene, NH). Bovine albumin (BSA)2 was obtained from the United States Biochemical Corp. (Cleveland, OH). Rabbit muscle actin was prepared by the method of Spudich and Watt (12). Site-specific polyclonal antibodies against actin were a gift from Dr. Gargi DasGupta and have been described previously (13). Alkaline phosphatase-conjugated goat anti-rabbit IgG was bought from Sigma Chemical Co. (St. Louis, MO). ScintiVerse Bio-HP (SO-X-14) was from Fisher (Fairlawn, NJ). [14C]Formaldehyde (55 mCi/mmol) was procured from NEN Research Products, DuPont (Boston, MA). Bicinchoninit acid protein assay (BCA) was from Pierce Chemical Co. (Rockford, IL). Coomassie brilliant blue R-250 was obtained from Schwarz/Mann Biotech (Cleveland, OH). Naphthol blue black and the Bradford protein assay reagent were purchased from Bio-Rad (Richmond, CA). Household 3-in-one lubricating oil was from Boyle-Midway, Inc. (New York, NY). Copper sulfate was Baker Analyzed, T. J. Baker, Inc. (Phillipsburg, NJ). All other reagents were of analytical grade. Protein Adsorption and Dot Blotting Proteins were diluted to appropriate concentrations in phosphate-buffered saline, pH 7.6 (PBS), and applied in 200 ~1 aliquots to 96-well Immulon 1 or 2 microtiter plates. Copper iodide staining determined that 4 h at 22°C gave maximal protein adsorption to plates (data not shown). Plates were then washed five times by submersion in deionized water and allowed to dry. Dot blotting was performed on nitrocellulose membranes which were soaked in PBS for 5 min and then allowed to dry. Proteins diluted in PBS were applied in 5-~1 drops and allowed to dry before staining.
REISLER
potassium iodide (20 g), sodium potassium tartrate (36 g), and 80 ml of deionized water were mixed into a fine slurry. Sodium hydroxide (10 g) was then dissolved into the solution which was subsequently allowed to stand at room temperature for 30 min. After a reddish precipitate settled, 70 ml of the supernatant was discarded. The remaining solvent and precipitate were mixed together prior to use. An excess of reagent was added to each microtiter plate well for an appropriate incubation time. The plate was then washed five times in deionized water and allowed to dry. Dot blots on nitrocellulose membranes were stained as previously described (1) except that 2-min incubations were used. Quantitation was performed with a Biomed Instruments (Fullerton, CA) Model SL-504-XL soft laser scanning densitometer equipped with an integrator and interfaced to an Apple IIe computer. Alternatively, a Dynatech (Chantilly, VA) MR600 microplate reader was used at a wavelength of 650 nm. The copper iodide stain was removed by filling the wells with 4 mM EDTA in PBS for 30 min. Plates were then washed with deionized water and allowed to dry. Coomassie and amido black staining reagents were prepared as described before (14). BCA protein assays were performed according to the manufacturer’s instructions and as originally described by Smith et al. (2). Enzyme immunoassays were performed by standard methods (15) but using 200-~1 aliquots per well. Radiolabeling and Counting BSA was labeled with 14Cby reductive methylation with [14C]formaldehyde (16). A 1 mg/ml BSA solution in 20 mM sodium cyanoborohydride and 100 mM Hepes, pH 9.0, was reacted for 24 h at 22°C with 6 mM [‘“Clformaldehyde (55 mCi/mmol). Three volumes of 10% trichloroacetic acid was added, and the precipitated protein was isolated by centrifugation. The pellet was washed once and redissolved in PBS. The protein concentration was determined by the BCA protein assay. The specific activity was determined to be 3.6 Ci/mmol by counting in 3 ml of ScintiVerse. [14C]BSA adsorbed to microtiter plates was quantified by excising the well with a hot knife (Cooper Tools, Apex, NC), dissolving the polystyrene overnight in ScintiVerse, and liquid scintillation counting (Beckman, LS 8100, Irvine, CA). Radiolabeled and native BSA had similar saturation plots on microtiter plates as judged by copper iodide staining (data not shown).
Staining and Enzyme Immunoassays The copper iodide staining reagent was prepared as described before (1). Copper sulfate pentahydrate (12 g), ’ Abbreviations used: BSA, bovine serum sity; PBS, phosphate-buffered saline; BCA, assay; IgG, immunoglobulin G.
albumin; OD, optical denbicinchoninic acid protein
Calibration of Microtiter
Plates
[14C]BSA was adsorbed to microtiter plate wells at desired concentrations and stained with copper iodide. [14C]BSA, between lo-100 ng/5 ~1 drop, was dot blotted onto a nitrocellulose membrane and stained with copper iodide. The dot blots were excised using a standard hole
COPPER
2.0
110
Incubation
3.0
IODIDE
4.0
STAINING
5.0
Time (min)
FIG. 1. Time course of copper iodide staining of microtiter plates. BSA (2 rg) was adsorbed to the microtiter plate, stained with copper iodide for the indicated periods of time, washed, and read with a densitometer. Bars represent the standard deviations (n = 3-4 wells).
puncher with a 6-mm diameter and placed into empty microtiter plate wells. Blank areas on the nitrocellulose membrane were punched out and used to cover wells with polystyrene-bound BSA that had been stained with copper iodide. Blank wells were also covered with blank nitrocellulose to measure the background. Five microliters of household 3-in-one lubricating oil was added to each well to render the nitrocellulose transparent (17). Staining was immediately quantified by densitometry and compared to results obtained by radioactivity measurements. RESULTS
AND
OF
MICROTITER
71
PLATES
a mixture of proteins within their range of independence allows all the proteins in the mixture to be represented on the plate in a consistent, although not necessarily stoichiometric, manner (4). A profile of the saturation curve (Fig. 2) enables the selection of optimal adsorption conditions and thus avoids considerable waste of the applied protein. To determine this profile accurately, the protein stain used must give a linear increase of density with the amount of protein present. To demonstrate the quantitative nature of copper iodide staining, the amount of absorbed protein was measured enzymatically and with [ ‘“C]BSA. Figure 3 shows good agreement between the alkaline phosphatase reaction and the copper iodide staining. The correlation coefficient was r2 = 0.982. Radioactivity measurements agreed even more closely with the densitometry of copper iodide staining as shown in Fig. 4. The correlation coefficient was r2 = 0.994. Thus, copper iodide staining is effective for quantitative measurements, and its density varies linearly with the amount of protein present. Since copper iodide staining is quantitative, the density (rig/mm’) of copper iodide-stained protein on a microtiter plate can be determined by the following procedure: (i) Dot blot the protein of interest in known amounts to form a standard curve. Typically lo-100 ng/ 5 ~1 drop, at least in quadruplicate, is dot blotted onto nitrocellulose and stained with copper iodide. (ii) Determine the average diameter of the dots and the average area covered by protein, typically 15.9 -t 1.1 mm2 (n = 43). (iii) Excise the dots with a hole puncher and place them in blank wells in the microtiter plate. To equalize
DISCUSSION
One of the attributes of copper iodide staining is its speed. Figure 1 shows a time course for copper iodide staining of BSA on microtiter plates. Maximal intensity of the stain was reached in 2-3 min; therefore, this incubation time was used for subsequent experiments. Incubations for up to 15 min did not result in any significant losses of staining intensity when compared to the plateau levels reached after 3 min of staining (data not shown). In principle, as shown in Fig. 2, densitometric and microtiter plate reader measurements of copper iodide staining can give similar results. The densitometric analysis, however, is more sensitive and yields more consistent results. Two possible reasons for the greater sensitivity of the densitometer are (i) a greater area of the microtiter plate well can be scanned and integrated, and (ii) the unfiltered densitometer integrates the entire visible adsorption spectrum of copper iodide, while the microtiter plate reader measures only a single wavelength. Figure 2 also demonstrates the range of independence of BSA adsorption. The value of 200 ng/200 ~1 found here is similar to the value of 1000 ng/lOOO ~1 found by Cantarero et al. (4). These authors found that adsorbing
120
-I
0
71
100
200
BSA
300
Applied
400
500
(ng)
FIG. 2. Comparison of densitometric and the microtiter plate reader measurements of copper iodide-stained protein. BSA was coated on to the microtiter plate wells at the indicated amounts per 200 ~1, stained with copper iodide, and read with both a densitometer and a microtiter plate reader. Open circles represent densitometer readings, while closed triangles are values from the microtiter plate reader (the maximum OD was 0.057 on the microtiter plate reader). Bars represent the standard deviations (n = 3 wells). Standard deviations for closed triangles are to the right of the symbol for clarity. Closed triangles have also been shifted to the right when they are at points identical with the circles.
72
ROOT
0
40
20 IgG
60 Applied
SO
AND
100
(ng)
the background, place blank nitrocellulose over both empty control wells and sample wells where the protein adsorbed directly to the microtiter plate has been stained with copper iodide. (iv) Add 5 ~1 of household 3in-one oil to render the nitrocellulose transparent and read with a densitometer.
60
o500
1000 BSA
Applied
0.0
2.0
4.0 BSA
FIG. 3. Comparison of copper iodide staining with enzymatic assays. Alkaline phosphatase-conjugated goat anti-rabbit IgG was adsorbed to the microtiter plates at the indicated amounts per 200 ~1, developed, and read like an enzyme immunoassay. The plate was then washed, stained with copper iodide, and read. Open circles represent copper iodide staining, while closed triangles are from enzymatic readings (the maximum OD was 1.368 on the microtiter plate reader). Bars represent standard deviations (n = 5 wells). Standard deviations for closed triangles are to the right of the symbol for clarity. Closed triangles have also been shifted to the right when they are at points identical with the circles.
0
REISLER
1500
2000
(ng)
FIG. 4. Comparison of copper iodide staining with radioactive assays. [%]BSA was adsorbed to microtiter plates at the amounts indicated per 200 ~1. The plate was stained with copper iodide and read. Then the wells were excised, and the amount of protein was determined by liquid scintillation counting. The lowest amount of protein detected by copper iodide staining was 3 ng per well. Open circles represent copper iodide staining, and closed triangles represent radioactive measurements (the maximum radioactivity was 280 Bq/well from [%]BSA with asp act of 3.6 Ci/mmol). Bars show the standard deviations (n = 5 wells). Standard deviations for closed triangles are to the right of the symbol for clarity. Closed triangles have also been shifted to the right when they are at points identical with the circles.
6.0
(rig/mm*)
FIG. 5. Calibration of microtiter plates by copper iodide staining. [‘%]BSA was adsorbed to microtiter plate wells (0,100,200,300,500, and 2000 ng/200 PI), washed, stained with copper iodide, and covered with blank nitrocellulose. Dot blots of [‘%]BSA (10,20,40,60,80, and 100 ng) were stained with copper iodide and placed in blank microtiter plate wells. Five microliters of household 3-in-one oil was added, and the plates were read by densitometry. The amount of protein coating the microtiter plate wells was verified by liquid scintillation counting. Densities were normalized to the area covered by protein. Open circles represent microtiter plate-bound protein (the maximum radioactivity of the open circles was 105 Bq/well from [14C]BSA with a sp act of 560 mCi/mmol), while closed circles represent nitrocellulose-bound protein. Bars show the standard deviations (n = 5-6).
From the density (rig/mm’) of protein on the microtiter plate, the total amount of adsorbed protein can then be calculated based on the dimensions of the well and the volume of protein solution originally added to it. For a 6.4-mm diameter flat-bottom well loaded with 200 ~1 of protein solution, the area covered by protein is 3.14r2 + (2V/r) = (3.14)(0.32 cm)’ + (2)(0.2 cm3)/0.32 cm = 1.57 cm’. Where r is the radius of the flat-bottom well, and V is the volume of the protein solution added for adsorption. This area can then be multiplied by the amount of protein per area determined from the standard curve of copper iodide-stained protein on nitrocellulose to render the total protein adsorbed to the microtiter plate. This method was used in Fig. 5 to determine the amounts of BSA adsorbed to a microtiter plate, and radioactivity measurements were used to verify the results. The accuracy of this method is based on two observations: (i) All of the protein applied to nitrocellulose by dot blots will be tightly bound, when saturation is not approached (M-21). (ii) Copper iodide staining did not cause a significant loss of protein from microtiter plates or nitrocellulose membranes as measured by radioactivity (data not shown). Correlation coefficients for radioactivity versus densitometric measurements of copper iodide-stained protein on nitrocellulose membranes or microtiter plates were r2 = 0.997 and r2 = 0.988, respectively. Thus, this copper iodide staining method of protein determination can substitute for radioactive measurements of exact amounts of bound protein.
COPPER
IODIDE
STAINING
The sensitivity of copper iodide staining on microtiter plates can be calculated from Fig. 4 as 20 pg/mm2. Silverenhanced colloidal gold may be more sensitive in detecting protein on microtiter plates (3), but its quantitative characteristics are more complex. In the case of horseradish peroxidase-conjugated IgG adsorbed to microtiter plates, the maximal intensity of silver-enhanced colloidal gold was reached at concentrations below enzymatitally detectable IgG (3). Copper iodide staining is, however, readily quantifiable in the same range of detection as enzyme assays (Fig. 3). Other attempts to detect and quantify proteins adsorbed to microtiter plates have had limited success. The BCA assay, like copper iodide, is copper-based (2), but it works only in solution and is not permanent (22). The BCA assay is less sensitive than copper iodide (22). In addition, it has not yet been shown that its extinction coefficients are the same for bound proteins as for proteins in solution (22). Ellipsometry has been used to detect adsorbed proteins (9,lO) but is also less sensitive than copper iodide staining and cannot be applied directly to microtiter plates. The Bradford protein assay, amido black, and Coomassie brilliant blue stains were unable to detect proteins adsorbed to microtiter plates (data not shown). Thus, copper iodide staining appears to be the most sensitive and proven method for estimating protein amounts on microtiter plates without the use of radioactivity. Copper iodide staining can be advantageous in comparative binding studies between different proteins. Take, as an example, an antibody being tested for its relative binding affinity to two different antigens. Microtiter plates could be coated with either of the two antigens, and the exact amount of adsorbed antigen could be assessed by copper iodide staining. A direct binding assay could then be normalized by the amount of antigen present. Such an experimental design would avoid problems of crosslinking by the antibody to both types of antigens which might occur in competitive binding assays. The reversibility of copper iodide staining of protein is another virtue of this procedure (1). A 30-min incubation with 4 mM EDTA in PBS is adequate to remove the stain from proteins on microtiter plates (data not shown). After actin was stained and destained with copper iodide, it was recognized by and would bind site-specific polyclonal antibody with an intensity similar to that of unstained actin (data not shown). Therefore, evaluation of the adsorbed antigen can be made both visually and quantitatively prior to reaction with the antibody. Copper iodide staining has many important attributes which make it valuable for use on microtiter plates. It
OF
MICROTITER
73
PLATES
is highly sensitive, quantitative, reversible, inexpensive, and convenient. In addition, the calibration technique presented here enables the accurate and sensitive determination of the amount of protein coating a microtiter plate. This procedure has many possible research applications particularly in the theory and practice of binding studies (6-11). Copper iodide staining may even be valuable for routine serological assays and quality control by visualizing the antigen coating at a lower cost than other techniques (3). ACKNOWLEDGMENT We thank to actin.
Dr. Gargi
DasGupta
for providing
site-specific
antibodies
REFERENCES 1. Root, D. D., and Reisler, E. (1989) Anal. &o&em. 181.250-253. 2. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gart ner, F. H., Provenzano, M. D., Fujimoto, N. M., Goeke, B. J., Olson, B. J., and Kenk, D. C. (1985) Anal. Biochem. 150,76-85. 3. Gibbs, J., and Root, D. M. (1988) Costar Research Newsletter, Vol. 1, Issue 2. 4. Cantarero, L. A., Butler, J. E., and Osborne, J. W. (1980) Anal. Biochem. 105,375-382. 5. Vogt, R. F., Phillips, D. L., Henderson, L. O., Whitfield, W., and Spierto, F. W. (1987) J. Immunol. Methods 101,43-50. 6. Fair, B. D., and Jamieson, A. M. (1980) J. Colloid Interface Sci. 77,525-534. I. Lee, R. G., Adamson, C., and Kim, S. W. (1974) Thromb. Res. 4, 485-490. 8. Nygren, H. (1988) J. Zmmunol. Methods 114,107-114. 9. Werthen, M., and Nygren, H. (1988) J. Immunol. Methods 115, ‘7-78. 10. Nygren, H., Kaartinen, M., and Stenberg, M. (1986) J. Zmmunol. Methods 92,219-225. 11. Stenberg, M., Werthen, M., Theander, S., and Nygren, H. (1988) J. Zmmunol. Methods 112,23-29. 12. Spudich, K., and Watt, S. (1971) J. Biol. Chem. 246,4866-4871. 13. Miller, L., Kalnoski, M., Yanossi, Z., Bulinski, J. C., and Reisler, E. (1987) Biochemistry 26,6064-6070. 14. Robyt, J. F., and White, B. J. (1987) Biochemical Techniques: Theory and Practice, pp. 144-145, Brooks/Cole Publishing Co., Monterey, CA. 15. Atherton, B. T., and Hynes, R. 0. (1981) Cell 25,133-141. 16. Jentoft, 4365. 17. Maciewicz,
N., and Dearborn,
D. G. (1979)
R. A., and Knight,
J. Biol
P. J. (1988)
Chem.
Anal.
254,4359-
Biochem.
175,
85-90. 18. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76.4350-4354. 19. Lin, W., and Kasamatsu, H. (1983) Anal. Biochem. 128,302-311. 20. Salinovich, O., and Montelaro, R. C. (1986) Anal. Biochem. 156, 341-347. 21. Enosawa, S., and Ohashi, A. (1987) Anal. Biochem. 160,211-216. 22. Sorensen, K., and Brodbeck, U. (1986) J. Immunol. Methods 95, 291-293.
