METHODS: A Companion to Methods in Enzymology 11, 419â425 (1997) ..... The easiest to prepare are samples from reticulocyte which leave a shadow on the ...
METHODS: A Companion to Methods in Enzymology 11, 419–425 (1997) Article No. ME960438
Use of Vertical Slab Isoelectric Focusing and Immunoblotting to Evaluate Steady-State Phosphorylation of eIF2a in Cultured Cells Olga Savinova and Rosemary Jagus1 Center of Marine Biotechnology, UMBI and Program in Oncology, University of Maryland Cancer Center, Baltimore, Maryland 21202
The combination of vertical, one-dimensional isoelectric focusing and immunoblotting works very well for the evaluation of the phosphorylation state of the a-subunit of eIF2 using reticulocyte lysate or purified eIF2. However, the method is more difficult to apply to the analysis of eIF2a phosphorylation in cultured cells. In part this reflects the fact that the protein content of cultured cell extracts is rarely as high as that found in extracts produced from reticulocytes, and in part this reflects the fact that some component(s) of cell extracts interferes with the entry of eIF2a into the isoelectric focusing gel. To overcome these difficulties, we have modified the earlier method to include immunoprecipitation of eIF2 from cell extracts prior to isoelectric focusing, as well as a low sodium dodecyl sulfate concentration in the isoelectric focusing sample buffer. Since the PKR activation state and therefore the eIF2a phosphorylation state change with cell density and nutritional status, we routinely set up consistent feeding schedules and recommend the collection of data over a range of cell densities. q 1997 Academic Press
Conditions for the evaluation of protein phosphorylation state using one-dimensional vertical slab iso1 To whom correspondence should be addressed at Center of Marine Biotechnology, Suite 236 Columbus Center, 701 E. Pratt Street, Baltimore, MD 21202. Fax: (410) 234-8896. E-mail: jagus @umbi.umd.edu.
electric focusing gels (VSIEF2 gels) followed by immunoblotting have previously been published (1). This method works particularly well for the analysis of eIF2a phosphorylation state in the reticulocyte cell-free translation system, as well as in kinase assays with purified components (2–4). It is more difficult to apply to the study of cultured cells. Part of the problem lies in the fact that extracts prepared from cells (1–10 mg/ml) do not reach the high protein concentration of reticulocyte lysate (Ç80 mg/ ml), giving a correspondingly lower concentration of eIF2. The problem of lower eIF2 levels in cell extracts is exacerbated by the fact that entry of proteins into an isoelectric focusing gel is not so efficient as entry into SDS–polyacrylamide gel. This is illustrated in Fig. 1, in which a comparison is made of the entry of eIF2a from extracts of reticulocytes and NIH 3T3 cells into SDS–polyacrylamide and VSIEF gels. eIF2 from reticulocyte lysate enters the VSIEF gel as efficiently as the SDS–polyacrylamide gel. However, although the eIF2a content of the NIH 3T3 sample is high enough to give a strong signal on immunoblots of the SDS–polyacrylamide gel, little is visible on immunoblots of the VSIEF gel. Treatment of cell extracts with ribonuclease or DNase has 2 Abbreviations used: Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; ECL, enhanced chemiluminescence; EDTA, ethylenediaminetetraacetic acid; eIF2, eukaryotic initiation factor-2; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; RRL, rabbit reticulocyte lysate; SDS, sodium dodecyl sulfate; VSIEF, vertical slab gel isoelectric focusing.
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1046-2023/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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no effect on this. However, there does seem to be some component(s) in the NIH 3T3 cell extract that actively interferes with entry of eIF2a into VSIEF gels. This is illustrated in Fig. 2, in which increasing amounts of NIH 3T3 cell extracts were added to reticulocyte lysate prior to sample preparation. As the amount of NIH 3T3 cell extract increases, less and less reticulocyte eIF2a enters the VSIEF gel. The approach we have taken to these problems is to immunoprecipitate eIF2 from cell extracts prior to fractionation by VSIEF.
DESCRIPTION OF THE METHOD Materials The materials used included the following: acrylamide, Polysciences, Warrington, PA; avidchrom-F Mab orientation gel, Unisyn Technologies, San Diego, CA; bisacrylamide, ammonium persulfate, and TEMED, Bio-Rad, Hercules, CA; BPA-1000, Toso-Haas, Philadelphia, PA; Chaps, Fluka, New York, NY; chymostatin, glutamic acid, b-glycerophosphate, histidine, and sodium molybdate, Sigma, St. Louis, MO; ECL kit and sheep anti-mouse IgG, peroxidase-linked, Amersham, Arlington Heights, IL; Elugent, Calbiochem, La Jolla, CA; ImmobilonP, Millipore, Bedford, MA; microcystin and versene, Gibco-Life Sciences, Grand Island, NY; PCC-54, Pierce, Rockford, IL; Pharmalytes, Pharmacia, Pis-
FIG. 1. Comparison of sample entry into SDS–polyacrylamide versus VSIEF gels. (A) Western blot of eIF2a from reticulocyte lysate (lanes 1 and 2) and NIH 3T3 cell extract (lanes 3 and 4), using a sample load of 25 mg (lanes 1 and 3) or 50 mg (lanes 2 and 4) separated by SDS–polyacrylamide gel electrophoresis. (B) Western blot of the same samples separated on VSIEF gels. The urea concentration was adjusted to 9.5 M in all samples before loading. Lanes 1 and 2 are reticulocyte lysate samples incubated with hemin and 2 mM EDTA (lane 1), or without hemin and 2 mM microcystin (lane 2), to indicate the positions of nonphosphorylated (lane 1) and phosphorylated (lane 2) eIF2a. Lanes 3 and 4 show reticulocyte lysate and lanes 5 and 6 show NIH 3T3 cell extracts loaded at the same levels as in A.
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cataway, NJ, or Sigma; urea (ultrapure), Fisher, Pittsburgh, PA. Care of Cells and Design of Experiments Figure 3 illustrates the changes in eIF2a phosphorylation state that occur with cell density and nutritional status of NIH 3T3 cells. As cell density increases, so does phosphorylation of eIF2a. Similarly, reducing the serum concentration from 10 to 2% increases eIF2a phosphorylation. If an investigator is looking at dramatic changes in eIF2a phosphorylation, as observed, for instance, in virus-infected cells, these possible variations in eIF2a phosphorylation state will probably not be a consideration (5). However, if an investigator is looking for more subtle changes, for instance the effects of a stably expressed foreign gene, it may be necessary to impose standard conditions on how cells are fed and used for experiments. Mid-log phase is ideal for most cell types, and the medium should be changed Ç20–24 h prior to harvest. When looking at the effects of the expression of nonfunctional PKR or PKR inhibitors on eIF2a phosphorylation, we changed the culture medium daily before harvest (6–8). Similarly, we always set up cultures at corresponding cell densities in 60-mm plates for cell density determination at time of harvest, harvesting samples at two or three different densities. In this way we were able to compare eIF2a phosphorylation in samples matched for cell density. Preparation of Cell Extracts for eIF2a Phosphorylation State Precautions must be taken to inactivate kinase and phosphatase activities during extract preparation. This is aided by the use of EDTA as a kinase
FIG. 2. A component of cell extracts prevents entry of eIF2a into VSIEF gels. The indicated amounts of reticulocyte lysate and NIH 3T3 cell extracts were incubated on ice for 3 min prior to the addition of VSIEF sample buffer and adjustment of the final urea sample to 9.5 M (lanes 3–9). Lanes 1 and 2 are reticulocyte lysate samples indicating the position of nonphosphorylated (lane 1) and phosphorylated (lane 2) eIF2a.
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inhibitor, microcystin (2 mM) as a phosphatase inhibitor in the lysis buffer, cold buffers, and speed. For cells growing as monolayers, we remove medium without cooling, place emptied plates on top of metal blocks sitting in ice, and rinse twice with ice-cold PBS containing 90 mM NaF, 17.5 mM sodium molybdate, and 17.5 mM b-glycerophosphate. In addition, we only process four plates at a time. After removal of all surplus PBS, we lyse cells from a 100-mm plate in 500 ml lysis buffer (20 mM Hepes-KOH, pH 7.2; 5 mM EDTA; 100 mM KCl; 0.005% SDS; 0.5% Elugent; 10% glycerol; 20 mg/ml chymostatin; 2 mM microcystin). The cell suspension is scraped into a microfuge tube and vortexed briefly. Five minutes is allowed to elapse between adding lysis buffer and beginning the microcentrifugation at 10,000g, at 47C, for 5 min. The supernatant is decanted carefully into a clean microfuge tube to which 5 ml BPA-1000 is added, followed by vortexing and microcentrifugation at 10,000g, at 47C, for 1 min. BPA-1000 removes some of the material that prevents eIF2a from entering the VSIEF gel. The clarified supernatant is decanted carefully into a clean microfuge tube, and a 20 mlaliquot is removed for protein determination. Depending on the cell type, the protein concentration will be between 1 and 10 mg/ml. The sample is snapfrozen in a metal block on dry ice and stored at 0707C. Figure 4 compares three variations in the preparation of cell extracts from NIH 3T3 cells, reflecting common laboratory practice. Lane 3 illustrates eIF2a phosphorylation from NIH 3T3 cells lysed under the conditions described above. Lane 4 shows the effects of sample preparation without the use of the phosphatase inhibitors in PBS and with sample processing slowed down by simultaneous processing of nine additional 100-mm plates. Loss of the phosphorylated form of eIF2a can be observed. Lane 5 shows the effects of scraping the cell layer in the
FIG. 3. Effects of cell density and serum concentration on eIF2a phosphorylation. Samples were prepared as described from NIH 3T3 cells after 1, 2, 3, 4 and 6 days in culture (DMEM, 10% calf serum), with daily medium changes. On Days 1 and 3, some plates of cells were shifted into 2% serum 24 h before samples were prepared on Days 2 and 4.
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presence of ice-cold PBS supplemented with phosphatase inhibitors and concentrating by microcentrifugation prior to lysis. Again, loss of the phosphorylated form of eIF2a can be observed. This modification is often considered when the analysis of eIF2a phosphorylation from slowly or sparsely growing cells is attempted, as a way of increasing the protein concentration of the extract. A preferable alternative can be seen in lane 6, in which cells are dissociated by incubation in versene (0.53 mM EDTA in PBS), at 47C, for 5 min, and concentrated by centrifugation at 300g, at 47C, for 5 min prior to rinsing twice in PBS supplemented with phosphatase inhibitors prior to lysis and processing. The phosphorylation state of eIF2a remains intact, making this the recommended procedure for sparse or slowly growing cells, since the volume of the lysis buffer can be reduced. Immunoprecipitation To concentrate eIF2, and remove it from the lysate contaminants that prevent entry into the VSIEF gel, we use monoclonal antibodies to eIF2a bound to beads. We use Unisyn’s Avidchrom Mab orientation gel (F), also called Hydrazide AvidGel F, and buy the bulk gel, using it with a coupling procedure supplied by the manufacturer. Briefly, the antibody is dialyzed against sodium acetate buffer, pH 5.0, overnight at 47C, and oxidized by exposure to 0.01 M sodium periodate, at pH 5.0, and 47C, in a lightproof container. The sodium periodate is removed by
FIG. 4. Comparison of lysis procedures. Lane 3 shows a sample prepared from NIH 3T3 cells by the standard method described under Description of the Method. (Lane 4) Sample preparation without the use of phosphatase inhibitors in PBS and with sample processing slowed down by the simultaneous processing of nine additional 100-mm plates. (Lane 5) Samples prepared by scraping cells from plate in ice-cold PBS supplemented with phosphatase inhibitors and concentration of cells by microcentrifugation prior to lysis. (Lane 6) Sample prepared by dissociation of cells in versene (5 mM EDTA in PBS) for 5 min at 47C, harvested by centrifugation at 300g for 5 min at 47C, and rinsed twice with ice-cold PBS supplemented with phosphatase inhibitors prior to lysis. (Lanes 1 and 2) Reticulocyte lysate samples indicating the position of nonphosphorylated (lane 1) and phosphorylated (lane 2) eIF2a.
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dialysis and the oxidized antibody is coupled to the gel in sodium acetate buffer, pH 5.0, for 16 h at 47C. We use Ç0.5 mg antibody per 1 ml resin. The antibody-linked resin is stored at 47C as a 1:1 suspension in PBS, 0.02% sodium azide, and rinsed three times with rinsing buffer (100 mM KCl; 20 mM Hepes-KOH, pH 7.2; 10% glycerol) prior to use. For immunoprecipitation, we use cell lysate equivalent to 250 mg of protein (usually 20–200 ml extract), bring samples up to an equal volume with lysis buffer, and add 400 ml binding buffer (100 mM KCl; 20 mM Hepes-KOH, pH 7.2; 0.4 mM EDTA; 10% glycerol; 20 mg/ml chymostatin; 0.5 mM microcystin), along with 20 ml beads (as a 1:1 suspension). The mixture is rocked at 47C for 6 h. The beads are collected by microcentrifugation at 10,000g at 47C for 5 min. The supernatant is discarded and the beads are rinsed with 1 ml of rinsing buffer (100 mM KCl; 20 mM Hepes-KOH, pH 7.2; 10% glycerol) prior to a final microcentrifugation at 10,000g at 47C for 5 min. Seventy microliters of VSIEF sample buffer (9.5 M urea; 1% Pharmalyte, pH 4.5–5.4; 1% Pharmalyte, pH 5–6; 0.15% SDS; 50 mM DTT) are added to the beads, which are incubated at 307C for 5 min to dissociate eIF2a from the beads. The volume of beads used (20 ml) has been determined to be optimal by testing a range of bead volumes against 250 mg protein (data not shown). Figure 5 shows the recovery of eIF2a from different input levels of cell extract, using 20 ml beads, showing proportional signal. Recovery of eIF2a is only about 20%. However, recovery is the same over the protein level tested (50–300 mg) and is the same for phosphorylated and nonphosphorylated forms of eIF2a (data not shown). Difficulties with Sample Entry into VSIEF Gel In attempting to overcome the problems of eIF2a entry into VSIEF gels, we compared immunoprecipi-
FIG. 5. Recovery of eIF2a from different input levels of cell extract. eIF2a from NIH 3T3 cell extracts equivalent to 50, 100, 200, and 300 mg protein was immunoprecipitated as described and subjected to SDS–polyacrylamide gel electrophoresis at 12.5% polyacrylamide (23). The immunoblot is shown here.
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tation with precipitation by trichloracetic acid (10%) or acetone. A comparison of these three methods is shown in Fig. 6A. Clearly, immunoprecipitation is the preferred method. The inclusion of 0.15% SDS in the sample buffer also improves eIF2a entry (data not shown), probably by improving the removal of eIF2a from the beads. The inclusion of SDS is not a problem since it is focused away from eIF2a during electrophoresis. However, as shown in Fig. 6B, the use of higher concentrations of SDS (1.5%) gives a lower signal, probably because highly charged eIF2a does not enter the gel. Theoretically, this could be circumvented by the use of normal polarity and the use of glutamic acid and histidine running buffers at the cathode and anode, respectively (the opposite of our usual running conditions). However, we have never found this to give us acceptable results with eIF2a. More eIF2a can be eluted by heating the beads at 957C for 5 min. However, heating is not a practicable procedure, as shown in Fig. 6C. A ladder of eIF2a bands appear, generated by carbamylation of the protein caused by the breakdown of urea. This result can also occur, to a lesser degree, by prolonged heating of the sample at temperatures higher than 307C (results not shown). Vertical Slab Gel Isoelectric Focusing This technique is sensitive to operator sloppiness and the procedure should be followed carefully. Over the years we have introduced many time-saving variations (intentional or otherwise), but always return to the method below. We use a stock solution of acrylamide/bisacrylamide, of 28.38 and 1.62%, respectively. This is stored at 47C in a light-proof container. The zwitterionic detergent, Chaps, is bought only from FLUKA. Chaps is made from cholate and batches from other sources have been found to be contaminated to varying degrees with this, giving great variability in results. Chaps is made up as a 20% stock solution and stored at 47C. The sample buffer (9.5 M urea; 1% Pharmalyte, pH 4.5–5.4; 1% Pharmalyte, pH 5–6; 0.15% SDS; 5% Chaps; 50 mM DTT) needs optimal operator attentiveness to make up. We use water prewarmed to 307C and try to resist adding too much water initially. For 25 ml of sample buffer, we put 14.26 g urea into a cylinder, add 6.25 ml 20% Chaps, bring the volume up to 23 ml with water, and incubate in a 307C water bath, stirring intermittently on a magnetic stirrer. After the urea is completely dis-
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solved, we add 192.5 mg DTT, 250 ml Pharmalytes, pH 4.5–5.4, 250 ml Pharmalytes, pH 5–6, 37.5 mg SDS, adjusting the final volume to 25 ml with water at 307C. One-milliliter aliquots should be snap-frozen, moving quickly before the urea comes out of solution, and stored at 0207C. Each aliquot should be used only once and discarded. Only electrophoresis grade urea should be used. Urea solutions should never be warmed to temperatures higher than 307C, since urea breakdown products can carbamylate proteins. A sample overlay containing 5 M urea and 1% of each Pharmalyte is also made up and stored similarly. Preparation of Gels For two vertical slab gels of 0.75 mm, such as the Hoefer SE600 apparatus, mix 25.34 g urea, 6 ml acrylamide/bisacrylamide stock solution, and 3 ml 20% Chaps with 14.55 ml water (at 307C) in a small glass beaker containing a magnetic stir bar. Alternate the beaker between a 307C waterbath and a magnetic stirrer until the urea is dissolved. Add 1.2 ml of each Pharmalyte, mix thoroughly, and immediately add 120 ml 10% ammonium persulfate (make fresh, with ice-cold water, for every use) and 48 ml TEMED. This gives final concentrations of 8.8 M
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urea, 3.75% acrylamide, 1.25% Chaps, 5% Pharmalytes, 0.025% ammonium persulfate. Mix well, pour into gel apparatus, add comb, and allow to polymerize for 30 min. When polymerized, remove comb and wash wells to remove any crystallized urea and drain very carefully to leave dry wells. We recommend not using the first or last two wells for sample, but instead loading them with sample buffer and overlay only. Samples are loaded into dry wells (20– 60 ml) and overlaid with enough sample overlay to fill the well. We use reversed polarity, with 0.01 M glutamic acid at the anode (top) and 0.05 M histidine at the cathode (bottom). The lower reservoir is completely filled with 0.05 M histidine to facilitate cooling. The gels are run at 2 mA per gel, with an 1200 voltage limit for 18 h (11,000 volt h). The lower buffer temperature is maintained at 187C by using a cooling baffle hooked up to a refrigerated recirculating pump. For urea gels, using tap water for cooling can be unsatisfactory because the urea falls out of solution if the temperature falls below 15/167C. In addition, a major source of variability in isoelectric focusing can be temperature variations (9). Control Samples To check technique, it is a good idea to run samples of which you know the status of phosphorylation.
FIG. 6. Effects of sample treatment on entry of eIF2a into VSIEF gels. (A) Precipitation by 10% trichloracetic acid (lanes 3–5), acetone (lanes 6–8), or immunoprecipitation (lanes 9–11) of NIH 3T3 cell extract equivalent to 62.5 (lanes 3, 6, 9), 125 (lanes 4, 7, 10), or 250 mg protein (lanes 5, 8, 11). (B) Comparison of entry of eIF2a from reticulocyte lysate (lanes 3, 4, 7, 8) or NIH 3T3 cell extracts (lanes 5, 6, 9, 10) into the VSIEF gel in samples prepared in VSIEF sample buffer containing 0.15 or 1.5% SDS. (C) Effects of heating a sample of reticulocyte lysate in VSIEF sample buffer at 957C for 5 min. In all panels, lanes 1 and 2 are reticulocyte lysate samples indicating the position of nonphosphorylated (lane 1) and phosphorylated (lane 2) eIF2a. Similarly, all panels show the immunoblots of the VSIEF gels.
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The easiest to prepare are samples from reticulocyte lysate incubated with hemin and 2 mM EDTA for 5 min at 307C, or minus hemin for 10 min, followed by a further 5 min in the presence of 1 mM microcystin. This will give samples with completely unphosphorylated or completely phosphorylated eIF2a, respectively (as illustrated in many of the figures in this paper). One microliter of RRL or MDL in 100 ml sample buffer works well, 20 ml of which gives a clear signal following immunoblotting and probing of the VSIEF gel. For controls of cell samples, we use interferon-treated HeLa cells incubated with or without poly(I):poly(C) (10–100 mg/ml) for 8–20 h or NIH 3T3 cells incubated with or without A23187, at 1 mM, for 15 min (10, 11). Transfer/Immunoblotting These gels have little integrity and should not be handled directly. We handle them much like sequencing gels and ensure that the gel plates are scrupulously clean. We soak used gel plates in wash gel plates in PCC-54 (Pierce), wash with acetone, then Liquinox, followed by Windex, rinsing well between each step with tap water, and with a final rinse in MilliQ water. To assemble a transfer cassette, we recommend removing one gel plate, placing a piece of prewetted Immobilon-P on top of the gel, and reversing them on top of the open transfer cassette. It is then possible to peel away the glass plate from the gel/Immobilon-P (as long as the gel plates are squeaky clean) before completing the assembly of the transfer cassette. We transfer at 60 V (0.4 A) for 1 h, using 250 mM glycine; 25 mM Tris, pH 8.3; 20% methanol; 0.01% SDS (NB. for SDS–polyacrylamide gels with higher polyacrylamide concentrations we transfer for 75 min). To keep the temperature down we mix our 51 transfer buffer stock (stored at 47C) and methanol with water at 47C. We find this preferable to using the cooling baffles,
FIG. 7. Analysis of eIF2a in a variety of cell lines. eIF2a from extracts equivalent to 250 mg protein was immunoprecipitated by the described protocol and subjected to VSIEF and immunoblotting. (Lanes 3–7) NIH 3T3, 293, HeLa, T47D, and Hs578B cells, respectively.
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which leave a shadow on the blot. After transfer, nonspecific sites on the Immobilon-P blots are blocked with BLOTTO (5% w/v nonfat dried milk; 0.01% Antifoam A; 200 mM NaCl; 50 mM Tris–HCl, pH 7.4) (12, 13) and probed with monoclonal antibodies to eIF2a, supplied by the laboratory of the late Dr. Ed Henshaw (14). Sheep anti-mouse IgG, linked to horseradish peroxidase, is used as the second antibody. All antibody incubations are done in BLOTTO at 47C, with shaking. Phosphorylated and nonphosphorylated eIF2a are visualized by enhanced chemiluminescence from the horseradish peroxidase catalyzed oxidation of luminol, using Amersham’s ECL reagents, as directed by the manufacturer. We always check the effectiveness of the immunoblotting reagents by including a narrow (2.5 mm) immunoblot from HeLa cell extracts for checking the eIF2a antibody and a narrow immunoblot from mouse serum for checking the second antibody. Using these two immunoblots also enables assessment of the ECL reagents. The narrow immunoblots are made by fractionating 1 mg of either HeLa cell extracts or mouse serum by SDS–polyacrylamide gel electrophoresis using a flat gel surface instead of wells and transferring to Immobilon-P. Troubleshooting Gels do not polymerize: Ammonium persulfate solution too old. Wavy bands: (a) Ammonium persulfate solution not fresh; (b) reservoir buffers not freshly made; (c) no cooling of gel during electrophoresis. Phosphorylated and nonphosphorylated forms of eIF2a do not separate: (a) Electrophoresis conditions not appropriate, e.g., too few volt hours; (b) too little urea or DTT in sample buffer; (c) old reservoir buffers; (d) Pharmalytes not stored appropriately. Low signal: Check immunoblotting reagents. Empty patches on blot: Uneven transfer, bubbles between gel and Immobilon-P. eIF2a cannot be seen: (a) Poor recovery of eIF2 in cell lysate; check by immunoblotting samples separated on SDS–polyacrylamide gels; (b) poor entry of sample into the VSIEF gel; try immunoprecipitating higher amount of sample; try reducing lysis volume. Extra bands: (a) Carbamylation from breakdown of urea probably caused by heating over 307C or inappropriate preparation or storage of VSIEF sample buffer; (b) Proteolysis; check protease inhibitors. Extra band near bottom of gel: Incomplete dissociation of eIF2a from b-subunit.
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ACKNOWLEDGMENTS
CONCLUDING REMARKS The procedure, essentially as described here, has been used in several investigations (5, 7, 8, 15–17) with a variety of cell types. In addition to reticulocyte lysate and the cell lines in these papers, we have shown the method to work for a variety of other cell lines, including HeLa, 293 cells, a normal breast, and a breast carcinoma cell line, as shown in Fig. 7. In addition, we have used it to look at eIF2a phosphorylation in a hematopoietic cell line (18), normal and transformed rat embryo fibroblasts, an ovarian carcinoma cell line, and a smooth muscle cell line (unpublished results). This method can also be adapted for use with other proteins with isoelectric points between pH 4 and pH 7. For instance, we have used it successfully for evaluating the phosphorylation state of eIF4E. For eIF4E, with pIs of 5.9 and 6.1 for the nonphosphorylated and phosphorylated forms, respectively, we use a mixture of Pharmalytes, 5–8 and 4–6.5 (0.5:1), along with 0.1 M glutamic acid at the anode (bottom) and 0.02 M NaOH at the cathode (top) (19). In addition, a variation of the method has been successfully used for the assessment of the phosphorylation state of elongation factor-2 (20). Resolution of basic proteins is more difficult, even with the use of nonequilibrium pH gradient electrophoresis, although a simple faster variation that could be adapted to VSIEF has recently been published (21). The procedure is not yet perfect and continues to be problematic for the analysis of eIF2a phosphorylation in cell lines that grow to low saturation densities or have low protein:DNA ratios. In particular, we are looking for better ways to prepare extracts so that immunoprecipitation is not necessary. A promising procedure has recently been published (22) and we are in the process of trying it out. Alternatively, it might be possible to develop a monoclonal antibody specific for the phosphorylated form of eIF2a to allow an assessment of phosphorylation state by screening the amounts of material interacting with each antibody on immunoblots from SDS–polyacrylamide gels or by a micro-ELISA technique.
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Thanks are extended to Dr. Bhavesh Joshi for critical review of the manuscript. This work was supported by DOD: DAMD1794-4324 and NIH: 1 RO1 CA67382.
REFERENCES 1. Maurides, P. M., Akkaraju, G. R., and Jagus, R. (1989) Anal. Biochem. 183, 144–151. 2. Jagus, R., and Safer, B. (1987) Adv. Cyclic Nucleotide Protein Phosphorylation Res. 11, 557–570. 3. Akkaraju, G. R., Whitaker-Dowling, P., Youngner, J. S., and Jagus, R. (1989) J. Biol. Chem. 264, 10321–10325. 4. Carroll, K., Elroy-Stein, O., Moss, B., and Jagus, R. (1993) J. Biol. Chem. 268, 12837–12842. 5. Swaminathan, S., Rajan, P., Savinova, O., Jagus, R., and Thimmapaya, B. (1996) Virology 219, 321–323. 6. Barber, G. N., Thompson, S., Lee, T. G., Strom, T., Jagus, R., Darveau, A., and Katze, M. G. (1994) Proc. Natl. Acad. Sci. USA 91, 4278–4282. 7. Barber, G., Wambach, M., Thompson, S., Jagus, R., and Katze, M. G. (1995) Mol. Cell. Biol. 15, 3138–3146. 8. Barber, G. N., Jagus, R., Meurs, E. F., Hovanessian, A., and Katze, M. G. (1995) J. Biol. Chem. 270, 17423–17428. 9. Gorg, A., Postel, W., Friedrich, C., Kuick, R., Strahler, J. R., and Hanash, S. M. (1991) Electrophoresis 12, 653–658. 10. Prostko, C. R., Dholakia, J. N., Brostrom, M. A., and Brostrom, C. O. (1995) J. Biol. Chem. 270, 6211–6215. 11. Srivastava, S. P., Davies, M. V., and Kaufman, R. J. (1995) J. Biol. Chem. 270, 16619–16624. 12. Johnson, G. A., Gautsch, J. W., Sportsman, J. R., and Elder, J. H. (1984) Gene Anal. Tech. 1, 3–8. 13. Jagus, R., and Pollard, J. W. (1988) Methods Mol. Biol. 3, 403–408. 14. Scorsone, K. A., Panniers, R., Rowlands, A. G., and Henshaw, E. C. (1987) J. Biol. Chem. 262, 14538–14543. 15. Donze, O., Jagus, R., Koromilas, A. E., Hershey, W. B., and Sonenberg, N. (1995) EMBO J. 14, 3828–3834. 16. Koromilas, A., Cantin, C., Craig, A., Jagus, R., Hiscott, J., and Sonenberg, N. (1995) J. Biol. Chem. 270, 25426–25434. 17. Offerman, M. K., Zimring, J., Mellits, K. H., Hagan, M. K., Shaw, R., Medford, R. M., Mathews, M. B., Goodbourn, S., and Jagus, R. (1995) Eur. J. Biochem. 232, 28–36. 18. Ito, T., Jagus, R., and May, S. (1994) Proc. Natl. Acad. Sci. USA 91, 7455–7459. 19. Jagus, R., Huang, W.-I., Hiremath, L. S., Stern, B. D., and Rhoads, R. E. (1993) Dev. Genet. 14, 412–423. 20. Redpath, N. T. (1992) Anal. Biochem. 202, 340–343. 21. Madden, M. S. (1995) Anal. Biochem. 229, 203–206. 22. Wang, K. K. W., Posner, A., and Hajimohammadreza, I. (1996) BioTechniques 20, 662–668. 23. Jagus, R. (1987) Methods Enzymol. 152, 296–306.
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