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[9]
tied by standard techniques including fast protein liquid chromatography (FPLC), glycerol gradient ultracentrifugation, and one- and two-dimensional SDS-PAGE. The eluted PY proteins are typically nondenatured, and thus can be analyzed for a variety of functional activities. These proteins can be stored at - 7 0 ° for >1 year with no obvious changes in their SDS electrophoretic properties. We have used this procedure with good success for isolating PY proteins from chronic myelogenous leukemia cells and, with minor modifications, for isolating PY proteins from growth factor-stimulated adherent cells. Greater than 90% of the protein phosphotyrosine in EGF-stimulated cells bound to the 1G2 immunosorbent (data not shown). The 1G2 hybridoma cells can be obtained from the ATCC (Rockville, MD) Safe Deposit collection, and the antibody itself is commercially available. Acknowledgment This w o r k was s u p p o r t e d in part by Grants R01-CA39235 and 5P30-CA13943 from the N a t i o n a l C a n c e r Institute.
[9] I s o l a t i o n o f T y r o s i n e - P h o s p h o r y l a t e d P r o t e i n s a n d Generation of Monoclonal Antibodies
By
JOHN R. GLENNEY
Introduction The mechanism by which tyrosine kinases exert their effects is not well understood. Although tyrosine-specific protein kinase activity was described more than 10 years ago, the relevant substrates that mediate the effects of tyrosine phosphorylation have been elusive. One approach to identification of tyrosine kinase substrates has been to analyze the phosphoamino acid content of proteins that are thought to play a role in the process affected by the tyrosine kinase. This approach assumes that we can deduce the identity of the relevant substrates and simply test them for the presence of phosphotyrosine. It is quite possible that some mediators of the tyrosine kinase signal are novel proteins that have not been previously studied. To gain insight into the function and regulation of such substrates we have taken the approach of (I) isolation of tyrosine-phosphorylated proteins using immunoaffinity chromatography with a highaffinity monoclonal anti-phosphotyrosine antibody and (2) generation of METHODS IN ENZYMOLOGY, VOL. 201
Copyright © 1991by AcademicPress, Inc. All rights of reproductionin any form reserved.
[9]
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monoclonal antibodies to the polypeptide backbone of individual substrates. The antibodies to substrates can then be used to study the distribution, turnover, and phosphorylation of the protein in addition to cloning the cDNA encoding the protein from an expression library. In this chapter two protocols for the isolation of phosphotyrosine-containing proteins are described, one using harsh denaturing conditions [boiling sodium dodecyl sulfate (SDS)] for lysis and extraction, and the second using more gentle buffers. Also included are the generation and use of the anti-phosphotyrosine affinity column and convenient assays for monitoring the protein and phosphotyrosine content of the eluted fractions. Methods used for the monoclonal antibody production to generate antibodies to the polypeptide backbone of individual substrates 1 are described.
Purification of Phosphotyrosine-Containing Proteins from Tissue Culture Cells by Affinity Purification on Anti-Phosphotyrosine Monoclonal Antibody Columns
Isolation of Proteins under Nondenaturing Conditions Tissue culture cells are grown on plastic 150-mm tissue culture dishes to approximately 90% confluence. If the cells contain a constitutively active tyrosine kinase such as v-src, the cells are lysed directly. In the case of growth factor receptors such as the epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor, the kinase must be first activated by ligand. The growth factor-containing medium may be reused. Medium is removed and the plates placed in a cold room or refrigerator for 5 min prior to lysis of the cells. Six milliliters of ice-cold buffer A [10 mM imidazole, 0.5 M NaCI, 1% (w/v) Triton X-100, 0.2 mM sodium vanadate, 0.2 mM phenyl methyl sulfonyl fluoride (PMSF), 2 mM sodium azide, pH 7.3] is added to each plate. Generally 10 plates are lysed at a time and are then stacked on a shaker in the cold room and left to extract for 30 min with gentle swirling. Forty to 50 plates can be harvested conveniently within 10-15 min in this way. The viscous lysate is scraped into the tubes of a Beckman (Palo Alto, CA) 45TI rotor and centrifuged at 40,000 rpm for 1.5 hr at 4°. After centrifugation the supernatant is carefully removed and added to a 250-ml conical centrifuge bottle (Corning, Corning, NY) together with 1-2 ml of immobilized anti-phosphotyrosine antibody. [We use a monoclonal antibody, PY20, 2 coupled at a concentration of 10 mg antibody/ml Affi-Gel (Bio-Rad, Richmond, CA).] PY20 I j. R. Glenney, Jr. and L. Zokas, J. Cell Biol. 108, 2401 (1989). 2 j. R. Glenney, Jr., L. Zokas, and M. P. Kamps, J. Immunol. Methods 109, 277 (1988).
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[9]
Affi-Gel is available through ICN Biomedicals (Costa Mesa, CA; Cat. # 152-369). The tube is capped and shaken vigorously for 3-6 hr at 4 °. This is followed by centrifugation at 500 g for 5 min to collect the affinity matrix. The supernatant is carefully removed from the Affi-Gel pellet, and the pellet is resuspended in a small volume of buffer B (10 mM imidazole, 0.1 M NaC1, and 0.1% Triton X-100) and placed into a small chromatography column. The column is washed with a minimum of 20 column volumes of buffer B at 4° and the substrates then eluted with buffer B containing 5 mM phenyl phosphate. A low flow rate (1 ml/hr) during elution helps sharpen the peak of phosphotyrosine-containing protein. Fractions (0.5 ml) are monitored as described below.
Isolation of Phosphotyrosine-Containing Proteins under Denaturing Conditions In order to ensure complete solubilization of the phosphotyrosinecontaining proteins (since many are known to be in an insoluble cytoskeletal matrix), we use a method to isolate the proteins after solubilization in SDS. The SDS lysis also has the advantages of (1) inhibiting phosphatases that could lead to a reduced recovery of the phosphotyrosine-containing proteins on the affinity column and (2) inactivating tyrosine kinases that may phosphorylate substrates after cell lysis. Cells are grown as described above and, after removal of the medium, SDS lysis buffer [3 ml of 1% (w/v) SDS, 10 mM Tris, pH 7.0, kept at 100°] is added and swirled quickly to lyse the cells. After adding lysis buffer to five plates, the plates are then put in a microwave oven and further heated on a setting of high for 5 sec. The plates are then restacked in a different order and reheated in the oven for an additional 5 sec. The plates are removed and the lysis buffer is then swirled on the bottom of the plate for 5-10 sec. The plates are allowed to stand on the bench at a slight angle to allow liquid to drain to one side of the plate. The viscous lysate is scraped into the tubes of a Beckman 45 TI rotor and centrifuged at 40,000 rpm for 2 hr at 10-15 °. The supernatant is carefully separated from the translucent pellet and then used either directly for affinity chromatography or stored at - 7 0 ° . We find it convenient when working up several hundred plates of cells to grow up and lyse 50 plates at a time and to store the lysate until all of the plates have been harvested in this way. The lysate from 50 plates is diluted fivefold to a final concentration of 0.2% SDS, adjusted to 1% Triton X-100, 50 mM NaC1, 10 mM imidazole, 0.2 mM sodium vanadate, and 1 mM EDTA, pH 7.3, and added to a 250-ml conical centrifuge tube, to which is then added 1-3 ml of PY20 Affi-Gel/tube and shaken as described above. In this case the lysate can be shaken with the Affi-Gel
[9]
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overnight. The affinity resin is collected by centrifugation and resuspended in buffer C (10 mM imidazole, 50 mM NaCI, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) and loaded into a small chromatography column. The column is then washed with a minimum of 20 vol of buffer C and then 2-3 vol of buffer C (reducing the Triton X-100 to 0.1% and the SDS to 0.05%). The column is then eluted with the same low-detergent buffer with the addition of 5 mM phenyl phosphate. Fractions are collected (0.5 ml) and monitored as described below.
Assay of Affinity Column The column fractions eluted from the anti-phosphotyrosine column can be assayed in a variety of ways. The appropriate way will be dictated by the amount of protein from the column and the sensitivity needed. Since the elution buffer contains high concentrations of phenyl phosphate or phosphotyrosine in addition to detergents, monitoring the absorbance at 280 nm is not practical. We find that in a standard preparation from transformed chick fibroblasts in which 100 plates of cells are used, the overall yield is approximately 0.5 to 1.0 mg of phosphotyrosine-containing protein. With this amount of protein one can monitor by standard protein assays. We find it convenient to monitor using the BCA protein assay (Pierce, Rockford, IL) since detergent and phenyl phosphate do not interfere. The assay is performed in 96-well test plates with comparison to known bovine serum albumin (BSA) concentrations that have been made up and diluted in the exact buffer used for the elution of the protein from the PY20 column. An aliquot (10-15/zl) of each fraction is placed in the wells of a 96-well test plate, to which 200 ~1 of the BCA protein assay mix is added. The plate is incubated at 37° for 30-60 min. The absorbance at 570 nm is then monitored using a microplate reader. The absorbance or protein concentration of each fraction is then plotted as a function of the fraction number. As an alternative to this direct protein assay, fractions can be monitored by Western blots using an anti-phosphotyrosine antibody (either polyclonal or monoclonal) to assess the yield of individual phosphotyrosine-containing proteins. We have observed, for instance, that some proteins are eluted by phenyl phosphate from the PY20 column very early, whereas others trail off for some time. Some proteins, like calpactin I and map kinase, are poorly retained on the column. Thus, assaying by Western blotting would be important if one were interested in proteins with unusual properties. To assay by Western blotting, 15/.d of each fraction is mixed with 15/xl of 2 × SDS sample buffer and subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins can be stained
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with silver or transferred to either nitrocellulose or Immobilon (Millipore, Bedford, MA) for Western blotting. After blocking nonspecific sites with 3% BSA the blots are treated with anti-phosphotyrosine antibodies followed by an iodinated second antibody (see above and [10] in this volume for methods). The peak fractions assessed using the protein determination, silver staining, or Western blotting are pooled and can be concentrated using an Amicon (Danvers, MA) Centricon concentrator to approximately 0.5-1 mg protein/ml. Immunization of Mice and Production of Hybridomas Directed Against Individual Phosphotyrosine-Containing Proteins Immunization
Phosphotyrosine-containing protein (0.1-0.5 mg), isolated as described above and to be used to immunize BALB/c mice, is emulsified with Freund's complete adjuvant. We use multiple subcutaneous, intraperitoneal, and intramuscular immunizations. Second and third immunizations are performed at 1-month intervals using one-half the amount of antigen used in the first immunization and substituting Freund's incomplete adjuvant while maintaining the same routes of immunization. One week after the third immunization the mouse is immunized by injection of soluble phosphotyrosine-containing proteins in the tail vein. This is followed by another intravenous immunization 2-3 days later. For the final intravenous immunization we use the phosphotyrosine-containing proteins dialyzed for at least 4-5 days against phosphate-buffered saline (PBS) to reduce the detergent concentration. Intravenous (tail vein) immunizations are performed using a 27-gauge needle and 100-200 ~1 total volume containing 100-200 ~g protein. The fusion is performed 2 days after the last immunization. In some cases the final series of intravenous immunizations with soluble antigen will be lethal to the mouse, due to excessive detergent in the antigen preparation or anaphylactic shock. For this reason the parental myloma cell line is readied for immediate hybridoma production in case the mouse dies unexpectedly. Fusion
One to 2 days following the last immunization, the mouse is sacrificed and the spleen removed. A small amount of blood is routinely collected by aspiration after severing the jugular vein. This serum can be very useful in developing an assay for screening the hybridomas. The spleen cells are dissociated by first mincing the spleen with a razor blade and scissors and
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then passing it through a sterile 5-ml syringe with an 18-gauge needle 10-20 times. The cell suspension is placed in a sterile 15-ml centrifuge tube and allowed to settle for 2-3 min. The suspension of cells is carefully removed from the top, leaving the large clumps of tissue and cells at the bottom. These are discarded. Both the spleen cell suspension and the parental myeloma cells are then centrifuged. We routinely use the PAI myeloma cell line as fusion partner. For each fusion we use the cells from five or six 150-mm plates of subconfluent myeloma cells growing exponentially and subcultured daily. Within this range the exact number of either spleen or myeloma cells does not appear to be critical. The cells are collected by centrifugation at 1000 g for 5 min. The myeloma cells are mixed with the spleen cells in a 50-ml conical centrifuge tube and brought up to 50 ml with serum-free Dulbecco's modified Eagle's medium (DMEM) and centrifuged as above. The medium is carefully removed and 2-3 ml of fusion solution added. Fusion solution consists of 5 g Kodak (Rochester, NY) polyethylene glycol 1450 (PEG), 0.5 ml dimethyl sulfoxide (DMSO), and 5 ml phosphate-buffered saline adjusted to a pH of 8.0 and sterile filtered. The prewarmed fusion solution is added over 45 sec with tapping of the side of the tube every few seconds to resuspend the cells. The cell suspension is then diluted with 50 ml of prewarmed DMEM over a period of 90 sec and allowed to sit for a further 8 min at room temperature, followed by 2-3 min at 37°. The cells are collected by centrifugation and resuspended in DMEM containing I0% horse serum supplemented with HAT (hypoxanthine, aminopterin, thymidine), purchased as a 50 × stock solution (Sigma, St. Louis, MO). The cell suspension is directly plated into 96-well tissue culture plates. In most cases it is convenient to use l0 plates, to which 20% of the total cell suspension is dispersed, and another l0 plates for the dispersal of 80% of the cell suspension. The plates that have one to three growing hybridomas/well are used for assay. The plates are then placed back in a 37° incubator and allowed to remain undisturbed for 5-6 days.
Assay of Hybridomas First screen: E L I S A
Optimally one would want to test plates that have one to three growing hybridomas per well. Generally we assay for antibodies at 9-11 days following the fusion. The fastest growing hybridomas in most cases do not yield antibodies. Do not be impatient if some of the wells turn yellow due to overgrowth. It is important that the enzyme-linked immunosorbent assay (ELISA) be standardized several times using a single batch of phos-
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photyrosine-containing protein antigen prior to screening hybridomas, since there are only a few days to test and select cells for further studies. Both the serum obtained at the time of fusion (see above) and the immunizing antigen used to coat the plates are titered to develop the ELISA. As a general guide, the serum can be diluted 1/I000 and the amount of antigen on the plate varied from 1 ng to 1/zg/well. Immulon 4 plates (Dynatech, Baton Rouge, LA) are used and 75/xl of the antigen dilution in H20 is added to each well. The protein is dried on the bottom of the well, using a hair dryer pointed at the plate for - 1 hr, or simply by incubating the plates overnight (not stacked) under a fume hood that has good air flow. After the plates are dry the nonspecific protein-binding sites are blocked by the addition of 200/zl 5% (w/v) BSA in I0 mM Tris-HC1, pH 7.2 to 7.4, 100 mM NaCI to each well. The blocking is allowed to proceed at least 1 hr at 25 °, but the plates can be conveniently stored in blocking buffer at 4 ° for several weeks. After blocking, the plates are washed in ELISA wash buffer [10 mM Tris-HCl, pH 7.2, 100 mM NaCI, 0.05% (v/v) Tween 20] several times. Washes are accomplished by flicking the liquid from the plate into a sink and refilling using a 500-ml squirt bottle filled with ELISA wash buffer. Cultural supernatant (75/xl) is then added to each well using a multichannel pipette and sterile pipette tips under a laminar flow hood. Make sure that the designations of the culture plate and test plate are the same, including the orientation, to prevent later uncertainty in aligning positives from the ELISA with cultures in the tissue culture plate. The test plates are incubated in first antibody for 2 hr, washed five to six times as above and then in second antibody (goat antimouse peroxidase-conjugated IgG diluted 1/1000 in ELISA wash buffer containing 3% BSA) for 2 hr. After a final five or six washes the assay is developed with the addition of 150 t~l 50 mM sodium citrate (pH 5.5) containing 0.4 mg o-phenylenediamine (available in preweighed tablet form from Sigma) and 0.5/xl 30% HzOJml. The plates are shielded from sunlight and checked every 5 rain. The reaction is stopped before the background becomes excessive (the background can vary from plate to plate) by the addition of 50 ~1 4 N HzSO4/well. The color reaction can be quantified by an ELISA reader; however, judgments made by eye give the same results. To select positives from the culture plates, the test plate and culture plate are placed side by side and the wells that were positive in the test plate are indicated by marks on both the top and bottom of the culture plate. False positives are very common in the side and corner wells, so if there is an abundance of positives these wells could be avoided. Even if false positives are selected in this first round of screening they will be identified as such in the second round (see below) before cloning. One
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should make sure that there is enough antigen on hand to assay 10 plates at least two or three times. It is not unusual when testing the hybidomas to have all of the wells give a positive reaction in the first assay. This could be due to residual spleen cells that survived for the first 4-5 days and continue to produce IgG directed against substrates, or excessive sensitivity of the assay. In any case, if all wells are positive, feed the cells (75/xl medium) and repeat the assay the very next day. After 10-11 days in the well of a 96-well plate, cells will very shortly overgrow and die, so that decisions as to whether they are positive or negative need to be made as soon as possible after the assay. We use the ELISA to initially screen but not to make final decisions on whether to clone and pursue these antibodies.
Second Screen: Western Blot Select as many hybridomas as possible that show a positive reaction by ELISA and rescreen by Western blotting or immunoprecipitation. Many antibodies will not recognize a protein in a Western blot or immunoprecipitation, even though they react strongly by ELISA. Valuable time can be wasted in trying to determine the nature of the antigen in these instances. For this reason we use Western blotting as a routine part of our initial screening. Cells from 96-well plates are gently resuspended and transferred to 24-well plates where they are allowed to grow again in HAT medium for a few more days. Each plate is examined microscopically every day and culture supernatants are removed when the cells are just coming to confluence. One milliliter is removed and replaced with 1 ml of fresh medium. This culture medium is tested in a Western blot by one of two methods, depending on the number to be tested: 1. If 1-20 hybridomas are to be tested per day it is usually easier to test "strips" of the antigen displayed on a Western blot. A single well gel is constructed and the phosphotyrosine-containing protein resolved by S D S - P A G E and transferred to nitrocellulose or Immobilon. The nitrocellulose can then be blocked and cut into strips for use in a standard blotting assay. From a standard size gel, 30-50 strips can be made. 2. Alternatively, if many culture supernatants (20-100) are to be tested in a single day, we have found that a device in which 28 can be tested at a time is particularly useful (Immunetics, Cambridge, MA). This involves running the phosphotyrosine-containing protein on a minigel containing only one wide sample slot, transferring to nitrocellulose and, after blocking with a high-protein solution, placing the entire blot in a frame in which individual " c h a m b e r s " are formed on the surface of the nitrocellulose. Each device holds 2 blots, with a total capacity of 56 antibody solutions
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(only 50 ~1 of antibody solution is needed). The blot can be washed while in the frame, then removed from the frame and treated in secondary antibody. If multiple blots are used, these can all be treated in a common solution of secondary antibody. If using either strips or a frame, a positive antibody control should be included. Either a monoclonal antibody to a protein known to be phosphorylated on tyrosine in the system, or the serum from the mouse used for fusion (diluted 1/100-I/1000 as determined previously) can be used as a control. A negative control may also be required. In our experience, using proteins isolated in native form from vs r c - t r a n s f o r m e d chick fibroblasts, we were able to find approximately 200-300 antibodies which reacted with substrates by ELISA, and of these 50 reacted with substrates by Western blotting. Many of these reacted with the same protein and thus, by using Western blotting in the second stage assay, it is possible to focus on a small number of antibodies to each protein. Based on these assays a small number of hybridomas (
ANALYSIS OF PROTEIN PHOSPHORYLATION
[9]
tied by standard techniques including fast protein liquid chromatography (FPLC), glycerol gradient ultracentrifugation, and one- and two-dimensional SDS-PAGE. The eluted PY proteins are typically nondenatured, and thus can be analyzed for a variety of functional activities. These proteins can be stored at - 7 0 ° for >1 year with no obvious changes in their SDS electrophoretic properties. We have used this procedure with good success for isolating PY proteins from chronic myelogenous leukemia cells and, with minor modifications, for isolating PY proteins from growth factor-stimulated adherent cells. Greater than 90% of the protein phosphotyrosine in EGF-stimulated cells bound to the 1G2 immunosorbent (data not shown). The 1G2 hybridoma cells can be obtained from the ATCC (Rockville, MD) Safe Deposit collection, and the antibody itself is commercially available. Acknowledgment This w o r k was s u p p o r t e d in part by Grants R01-CA39235 and 5P30-CA13943 from the N a t i o n a l C a n c e r Institute.
[9] I s o l a t i o n o f T y r o s i n e - P h o s p h o r y l a t e d P r o t e i n s a n d Generation of Monoclonal Antibodies
By
JOHN R. GLENNEY
Introduction The mechanism by which tyrosine kinases exert their effects is not well understood. Although tyrosine-specific protein kinase activity was described more than 10 years ago, the relevant substrates that mediate the effects of tyrosine phosphorylation have been elusive. One approach to identification of tyrosine kinase substrates has been to analyze the phosphoamino acid content of proteins that are thought to play a role in the process affected by the tyrosine kinase. This approach assumes that we can deduce the identity of the relevant substrates and simply test them for the presence of phosphotyrosine. It is quite possible that some mediators of the tyrosine kinase signal are novel proteins that have not been previously studied. To gain insight into the function and regulation of such substrates we have taken the approach of (I) isolation of tyrosine-phosphorylated proteins using immunoaffinity chromatography with a highaffinity monoclonal anti-phosphotyrosine antibody and (2) generation of METHODS IN ENZYMOLOGY, VOL. 201
Copyright © 1991by AcademicPress, Inc. All rights of reproductionin any form reserved.
[9]
ISOLATION OF TYROSINE-PHOSPHORYLATED PROTEINS
93
monoclonal antibodies to the polypeptide backbone of individual substrates. The antibodies to substrates can then be used to study the distribution, turnover, and phosphorylation of the protein in addition to cloning the cDNA encoding the protein from an expression library. In this chapter two protocols for the isolation of phosphotyrosine-containing proteins are described, one using harsh denaturing conditions [boiling sodium dodecyl sulfate (SDS)] for lysis and extraction, and the second using more gentle buffers. Also included are the generation and use of the anti-phosphotyrosine affinity column and convenient assays for monitoring the protein and phosphotyrosine content of the eluted fractions. Methods used for the monoclonal antibody production to generate antibodies to the polypeptide backbone of individual substrates 1 are described.
Purification of Phosphotyrosine-Containing Proteins from Tissue Culture Cells by Affinity Purification on Anti-Phosphotyrosine Monoclonal Antibody Columns
Isolation of Proteins under Nondenaturing Conditions Tissue culture cells are grown on plastic 150-mm tissue culture dishes to approximately 90% confluence. If the cells contain a constitutively active tyrosine kinase such as v-src, the cells are lysed directly. In the case of growth factor receptors such as the epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor, the kinase must be first activated by ligand. The growth factor-containing medium may be reused. Medium is removed and the plates placed in a cold room or refrigerator for 5 min prior to lysis of the cells. Six milliliters of ice-cold buffer A [10 mM imidazole, 0.5 M NaCI, 1% (w/v) Triton X-100, 0.2 mM sodium vanadate, 0.2 mM phenyl methyl sulfonyl fluoride (PMSF), 2 mM sodium azide, pH 7.3] is added to each plate. Generally 10 plates are lysed at a time and are then stacked on a shaker in the cold room and left to extract for 30 min with gentle swirling. Forty to 50 plates can be harvested conveniently within 10-15 min in this way. The viscous lysate is scraped into the tubes of a Beckman (Palo Alto, CA) 45TI rotor and centrifuged at 40,000 rpm for 1.5 hr at 4°. After centrifugation the supernatant is carefully removed and added to a 250-ml conical centrifuge bottle (Corning, Corning, NY) together with 1-2 ml of immobilized anti-phosphotyrosine antibody. [We use a monoclonal antibody, PY20, 2 coupled at a concentration of 10 mg antibody/ml Affi-Gel (Bio-Rad, Richmond, CA).] PY20 I j. R. Glenney, Jr. and L. Zokas, J. Cell Biol. 108, 2401 (1989). 2 j. R. Glenney, Jr., L. Zokas, and M. P. Kamps, J. Immunol. Methods 109, 277 (1988).
94
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[9]
Affi-Gel is available through ICN Biomedicals (Costa Mesa, CA; Cat. # 152-369). The tube is capped and shaken vigorously for 3-6 hr at 4 °. This is followed by centrifugation at 500 g for 5 min to collect the affinity matrix. The supernatant is carefully removed from the Affi-Gel pellet, and the pellet is resuspended in a small volume of buffer B (10 mM imidazole, 0.1 M NaC1, and 0.1% Triton X-100) and placed into a small chromatography column. The column is washed with a minimum of 20 column volumes of buffer B at 4° and the substrates then eluted with buffer B containing 5 mM phenyl phosphate. A low flow rate (1 ml/hr) during elution helps sharpen the peak of phosphotyrosine-containing protein. Fractions (0.5 ml) are monitored as described below.
Isolation of Phosphotyrosine-Containing Proteins under Denaturing Conditions In order to ensure complete solubilization of the phosphotyrosinecontaining proteins (since many are known to be in an insoluble cytoskeletal matrix), we use a method to isolate the proteins after solubilization in SDS. The SDS lysis also has the advantages of (1) inhibiting phosphatases that could lead to a reduced recovery of the phosphotyrosine-containing proteins on the affinity column and (2) inactivating tyrosine kinases that may phosphorylate substrates after cell lysis. Cells are grown as described above and, after removal of the medium, SDS lysis buffer [3 ml of 1% (w/v) SDS, 10 mM Tris, pH 7.0, kept at 100°] is added and swirled quickly to lyse the cells. After adding lysis buffer to five plates, the plates are then put in a microwave oven and further heated on a setting of high for 5 sec. The plates are then restacked in a different order and reheated in the oven for an additional 5 sec. The plates are removed and the lysis buffer is then swirled on the bottom of the plate for 5-10 sec. The plates are allowed to stand on the bench at a slight angle to allow liquid to drain to one side of the plate. The viscous lysate is scraped into the tubes of a Beckman 45 TI rotor and centrifuged at 40,000 rpm for 2 hr at 10-15 °. The supernatant is carefully separated from the translucent pellet and then used either directly for affinity chromatography or stored at - 7 0 ° . We find it convenient when working up several hundred plates of cells to grow up and lyse 50 plates at a time and to store the lysate until all of the plates have been harvested in this way. The lysate from 50 plates is diluted fivefold to a final concentration of 0.2% SDS, adjusted to 1% Triton X-100, 50 mM NaC1, 10 mM imidazole, 0.2 mM sodium vanadate, and 1 mM EDTA, pH 7.3, and added to a 250-ml conical centrifuge tube, to which is then added 1-3 ml of PY20 Affi-Gel/tube and shaken as described above. In this case the lysate can be shaken with the Affi-Gel
[9]
I S O L A T I O N OF T Y R O S I N E - P H O S P H O R Y L A T E D PROTEINS
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overnight. The affinity resin is collected by centrifugation and resuspended in buffer C (10 mM imidazole, 50 mM NaCI, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) and loaded into a small chromatography column. The column is then washed with a minimum of 20 vol of buffer C and then 2-3 vol of buffer C (reducing the Triton X-100 to 0.1% and the SDS to 0.05%). The column is then eluted with the same low-detergent buffer with the addition of 5 mM phenyl phosphate. Fractions are collected (0.5 ml) and monitored as described below.
Assay of Affinity Column The column fractions eluted from the anti-phosphotyrosine column can be assayed in a variety of ways. The appropriate way will be dictated by the amount of protein from the column and the sensitivity needed. Since the elution buffer contains high concentrations of phenyl phosphate or phosphotyrosine in addition to detergents, monitoring the absorbance at 280 nm is not practical. We find that in a standard preparation from transformed chick fibroblasts in which 100 plates of cells are used, the overall yield is approximately 0.5 to 1.0 mg of phosphotyrosine-containing protein. With this amount of protein one can monitor by standard protein assays. We find it convenient to monitor using the BCA protein assay (Pierce, Rockford, IL) since detergent and phenyl phosphate do not interfere. The assay is performed in 96-well test plates with comparison to known bovine serum albumin (BSA) concentrations that have been made up and diluted in the exact buffer used for the elution of the protein from the PY20 column. An aliquot (10-15/zl) of each fraction is placed in the wells of a 96-well test plate, to which 200 ~1 of the BCA protein assay mix is added. The plate is incubated at 37° for 30-60 min. The absorbance at 570 nm is then monitored using a microplate reader. The absorbance or protein concentration of each fraction is then plotted as a function of the fraction number. As an alternative to this direct protein assay, fractions can be monitored by Western blots using an anti-phosphotyrosine antibody (either polyclonal or monoclonal) to assess the yield of individual phosphotyrosine-containing proteins. We have observed, for instance, that some proteins are eluted by phenyl phosphate from the PY20 column very early, whereas others trail off for some time. Some proteins, like calpactin I and map kinase, are poorly retained on the column. Thus, assaying by Western blotting would be important if one were interested in proteins with unusual properties. To assay by Western blotting, 15/.d of each fraction is mixed with 15/xl of 2 × SDS sample buffer and subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins can be stained
96
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with silver or transferred to either nitrocellulose or Immobilon (Millipore, Bedford, MA) for Western blotting. After blocking nonspecific sites with 3% BSA the blots are treated with anti-phosphotyrosine antibodies followed by an iodinated second antibody (see above and [10] in this volume for methods). The peak fractions assessed using the protein determination, silver staining, or Western blotting are pooled and can be concentrated using an Amicon (Danvers, MA) Centricon concentrator to approximately 0.5-1 mg protein/ml. Immunization of Mice and Production of Hybridomas Directed Against Individual Phosphotyrosine-Containing Proteins Immunization
Phosphotyrosine-containing protein (0.1-0.5 mg), isolated as described above and to be used to immunize BALB/c mice, is emulsified with Freund's complete adjuvant. We use multiple subcutaneous, intraperitoneal, and intramuscular immunizations. Second and third immunizations are performed at 1-month intervals using one-half the amount of antigen used in the first immunization and substituting Freund's incomplete adjuvant while maintaining the same routes of immunization. One week after the third immunization the mouse is immunized by injection of soluble phosphotyrosine-containing proteins in the tail vein. This is followed by another intravenous immunization 2-3 days later. For the final intravenous immunization we use the phosphotyrosine-containing proteins dialyzed for at least 4-5 days against phosphate-buffered saline (PBS) to reduce the detergent concentration. Intravenous (tail vein) immunizations are performed using a 27-gauge needle and 100-200 ~1 total volume containing 100-200 ~g protein. The fusion is performed 2 days after the last immunization. In some cases the final series of intravenous immunizations with soluble antigen will be lethal to the mouse, due to excessive detergent in the antigen preparation or anaphylactic shock. For this reason the parental myloma cell line is readied for immediate hybridoma production in case the mouse dies unexpectedly. Fusion
One to 2 days following the last immunization, the mouse is sacrificed and the spleen removed. A small amount of blood is routinely collected by aspiration after severing the jugular vein. This serum can be very useful in developing an assay for screening the hybridomas. The spleen cells are dissociated by first mincing the spleen with a razor blade and scissors and
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then passing it through a sterile 5-ml syringe with an 18-gauge needle 10-20 times. The cell suspension is placed in a sterile 15-ml centrifuge tube and allowed to settle for 2-3 min. The suspension of cells is carefully removed from the top, leaving the large clumps of tissue and cells at the bottom. These are discarded. Both the spleen cell suspension and the parental myeloma cells are then centrifuged. We routinely use the PAI myeloma cell line as fusion partner. For each fusion we use the cells from five or six 150-mm plates of subconfluent myeloma cells growing exponentially and subcultured daily. Within this range the exact number of either spleen or myeloma cells does not appear to be critical. The cells are collected by centrifugation at 1000 g for 5 min. The myeloma cells are mixed with the spleen cells in a 50-ml conical centrifuge tube and brought up to 50 ml with serum-free Dulbecco's modified Eagle's medium (DMEM) and centrifuged as above. The medium is carefully removed and 2-3 ml of fusion solution added. Fusion solution consists of 5 g Kodak (Rochester, NY) polyethylene glycol 1450 (PEG), 0.5 ml dimethyl sulfoxide (DMSO), and 5 ml phosphate-buffered saline adjusted to a pH of 8.0 and sterile filtered. The prewarmed fusion solution is added over 45 sec with tapping of the side of the tube every few seconds to resuspend the cells. The cell suspension is then diluted with 50 ml of prewarmed DMEM over a period of 90 sec and allowed to sit for a further 8 min at room temperature, followed by 2-3 min at 37°. The cells are collected by centrifugation and resuspended in DMEM containing I0% horse serum supplemented with HAT (hypoxanthine, aminopterin, thymidine), purchased as a 50 × stock solution (Sigma, St. Louis, MO). The cell suspension is directly plated into 96-well tissue culture plates. In most cases it is convenient to use l0 plates, to which 20% of the total cell suspension is dispersed, and another l0 plates for the dispersal of 80% of the cell suspension. The plates that have one to three growing hybridomas/well are used for assay. The plates are then placed back in a 37° incubator and allowed to remain undisturbed for 5-6 days.
Assay of Hybridomas First screen: E L I S A
Optimally one would want to test plates that have one to three growing hybridomas per well. Generally we assay for antibodies at 9-11 days following the fusion. The fastest growing hybridomas in most cases do not yield antibodies. Do not be impatient if some of the wells turn yellow due to overgrowth. It is important that the enzyme-linked immunosorbent assay (ELISA) be standardized several times using a single batch of phos-
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photyrosine-containing protein antigen prior to screening hybridomas, since there are only a few days to test and select cells for further studies. Both the serum obtained at the time of fusion (see above) and the immunizing antigen used to coat the plates are titered to develop the ELISA. As a general guide, the serum can be diluted 1/I000 and the amount of antigen on the plate varied from 1 ng to 1/zg/well. Immulon 4 plates (Dynatech, Baton Rouge, LA) are used and 75/xl of the antigen dilution in H20 is added to each well. The protein is dried on the bottom of the well, using a hair dryer pointed at the plate for - 1 hr, or simply by incubating the plates overnight (not stacked) under a fume hood that has good air flow. After the plates are dry the nonspecific protein-binding sites are blocked by the addition of 200/zl 5% (w/v) BSA in I0 mM Tris-HC1, pH 7.2 to 7.4, 100 mM NaCI to each well. The blocking is allowed to proceed at least 1 hr at 25 °, but the plates can be conveniently stored in blocking buffer at 4 ° for several weeks. After blocking, the plates are washed in ELISA wash buffer [10 mM Tris-HCl, pH 7.2, 100 mM NaCI, 0.05% (v/v) Tween 20] several times. Washes are accomplished by flicking the liquid from the plate into a sink and refilling using a 500-ml squirt bottle filled with ELISA wash buffer. Cultural supernatant (75/xl) is then added to each well using a multichannel pipette and sterile pipette tips under a laminar flow hood. Make sure that the designations of the culture plate and test plate are the same, including the orientation, to prevent later uncertainty in aligning positives from the ELISA with cultures in the tissue culture plate. The test plates are incubated in first antibody for 2 hr, washed five to six times as above and then in second antibody (goat antimouse peroxidase-conjugated IgG diluted 1/1000 in ELISA wash buffer containing 3% BSA) for 2 hr. After a final five or six washes the assay is developed with the addition of 150 t~l 50 mM sodium citrate (pH 5.5) containing 0.4 mg o-phenylenediamine (available in preweighed tablet form from Sigma) and 0.5/xl 30% HzOJml. The plates are shielded from sunlight and checked every 5 rain. The reaction is stopped before the background becomes excessive (the background can vary from plate to plate) by the addition of 50 ~1 4 N HzSO4/well. The color reaction can be quantified by an ELISA reader; however, judgments made by eye give the same results. To select positives from the culture plates, the test plate and culture plate are placed side by side and the wells that were positive in the test plate are indicated by marks on both the top and bottom of the culture plate. False positives are very common in the side and corner wells, so if there is an abundance of positives these wells could be avoided. Even if false positives are selected in this first round of screening they will be identified as such in the second round (see below) before cloning. One
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should make sure that there is enough antigen on hand to assay 10 plates at least two or three times. It is not unusual when testing the hybidomas to have all of the wells give a positive reaction in the first assay. This could be due to residual spleen cells that survived for the first 4-5 days and continue to produce IgG directed against substrates, or excessive sensitivity of the assay. In any case, if all wells are positive, feed the cells (75/xl medium) and repeat the assay the very next day. After 10-11 days in the well of a 96-well plate, cells will very shortly overgrow and die, so that decisions as to whether they are positive or negative need to be made as soon as possible after the assay. We use the ELISA to initially screen but not to make final decisions on whether to clone and pursue these antibodies.
Second Screen: Western Blot Select as many hybridomas as possible that show a positive reaction by ELISA and rescreen by Western blotting or immunoprecipitation. Many antibodies will not recognize a protein in a Western blot or immunoprecipitation, even though they react strongly by ELISA. Valuable time can be wasted in trying to determine the nature of the antigen in these instances. For this reason we use Western blotting as a routine part of our initial screening. Cells from 96-well plates are gently resuspended and transferred to 24-well plates where they are allowed to grow again in HAT medium for a few more days. Each plate is examined microscopically every day and culture supernatants are removed when the cells are just coming to confluence. One milliliter is removed and replaced with 1 ml of fresh medium. This culture medium is tested in a Western blot by one of two methods, depending on the number to be tested: 1. If 1-20 hybridomas are to be tested per day it is usually easier to test "strips" of the antigen displayed on a Western blot. A single well gel is constructed and the phosphotyrosine-containing protein resolved by S D S - P A G E and transferred to nitrocellulose or Immobilon. The nitrocellulose can then be blocked and cut into strips for use in a standard blotting assay. From a standard size gel, 30-50 strips can be made. 2. Alternatively, if many culture supernatants (20-100) are to be tested in a single day, we have found that a device in which 28 can be tested at a time is particularly useful (Immunetics, Cambridge, MA). This involves running the phosphotyrosine-containing protein on a minigel containing only one wide sample slot, transferring to nitrocellulose and, after blocking with a high-protein solution, placing the entire blot in a frame in which individual " c h a m b e r s " are formed on the surface of the nitrocellulose. Each device holds 2 blots, with a total capacity of 56 antibody solutions
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(only 50 ~1 of antibody solution is needed). The blot can be washed while in the frame, then removed from the frame and treated in secondary antibody. If multiple blots are used, these can all be treated in a common solution of secondary antibody. If using either strips or a frame, a positive antibody control should be included. Either a monoclonal antibody to a protein known to be phosphorylated on tyrosine in the system, or the serum from the mouse used for fusion (diluted 1/100-I/1000 as determined previously) can be used as a control. A negative control may also be required. In our experience, using proteins isolated in native form from vs r c - t r a n s f o r m e d chick fibroblasts, we were able to find approximately 200-300 antibodies which reacted with substrates by ELISA, and of these 50 reacted with substrates by Western blotting. Many of these reacted with the same protein and thus, by using Western blotting in the second stage assay, it is possible to focus on a small number of antibodies to each protein. Based on these assays a small number of hybridomas (
