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BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 100537 DESCRIPTION OF THE PROBLEM I ordered three antibodies, ab412, ab413, and ab414. I am currently trying to optimize the conditions for Western blotting using these antibodies. None of these antibodies are able to immunostain my protein of interest, which is ICP27 otherwise known as IE63 from the herpes simplex type 1 virus. Published literature shows that this protein is known to be methylated. SAMPLE A total of 2*10^7 HeLa cells were infected with wild-type HSV-1 virus. Cells were harvested 5 hours post-infection. Nuclear and cytoplasmic fractions were separated. Our protein of interest, ICP27, was immunoprecipitated with our monoconal antibody H1119. The immunoprecipitate was resolved using 12% SDS-PAGE, transferred o/n to nitrocellulose, and Western analysis was done on 4 parallel blots using our monoclonal antibody H1119, and ab 412, ab413, and ab414. I have tried running crude extracts on a 12% SDS-PAGE gel, transferring to nitrocellulose, and Western blotting using H1119, which is a monoclonal antibody toward ICP27, as well as ab412, ab413, and ab414. These were done on parallel blots. PRIMARY ANTIBODY mab 412 (abcam) mouse monoclonal IgG mab 413 (abcam) mouse monoclonal IgM mab 414 (abcam) mouse monoclonal IgG diluted at (see concentrations we have tried below) into 0.3%BSA/0.1% Tween/PBS incubate at room temp on rotator for 1 hour wash 15 min, 5 min, 5 min with 0.1%Tween/PBS add secondary antibody DETECTION METHOD SuperSignal West Pico chemiluminescent substrate from Pierce POSITIVE AND NEGATIVE CONTROLS USED + controls: we run crude extracts in parallel and detect a bunch of proteins. we also are sure that ICP27 is present in the extracts, because we run a parallel Western using a stellar monoclonal antibody against ICP27. We see ICP27 in all samples we test; however, your antibodies do not show staining to ICP27 even though we know it is methylated in vivo. ANTIBODY STORAGE CONDITIONS Anitbody was reconstituted in 200 uL ddH20. Separated into 40 uL aliquots and stored at -20C. SAMPLE PREPARATION Protease inhibitors used = leupeptin and pefabloc buffers = immunoprecipitates were washed in hi salt lysis buffer followed by PBS, SDS-PAGE loading buffer was added to immunopreciptates (+10% beta mercaptoethanol, and boiled 5 min. AMOUNT OF PROTEIN LOADED Crude extract: 5% of the total nuclear or cytoplasmic HeLa extract taken from 2*10^7 cells. IP: approx. 5-10 ug ICP27 per lane ELECTROPHORESIS/GEL CONDITIONS 12% SDS-PAGE TRANSFER AND BLOCKING CONDITIONS Transfer buffer: 300 mL 10X Tris-glycine 600 mL methanol 2100 mL ddH2O transfer o/n our MW markers are colored, so we know the transfer are successful when the markers transferred, this step is never a problem for us block in 5% BSA/0.1%Tween PBS 1 hr. wash 15 min, 5 min, 5 min in 0.1%Tween PBS 1 hr. add primary antibody SECONDARY ANTIBODY Manufacturer: [a competitor] Species: anti-mouse IgG (for H1119, ab412, ab414) anti-mouse IgM (for ab413) diluted at 1:100,000 into 0.3%BSA/0.1% Tween/PBS incubate at room temp on rotator for 1 hour wash 15 min, 5 min, 5 min with 0.1%Tween/PBS add ECL enhancer and substrate HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 WHAT STEPS HAVE YOU ALTERED? amount of primary antibody (ab 412, 413, 414) used in WB. Crude hela extracts (nuc & cyto fractionated): WB Attempt #1 ab412 1:5000 did not work ab413 1:500 did not work ab414 1:500 did not work WB Attempt #2 ab412 1:250 worked ab413 1:250 worked ab414 1:250 worked Immunoprecipitated ICP27 from HSV1-infected HeLa extracts WB Attempt#1 ab412 1:250 did not work, did not see ANY bands except heavy & light chains ab413 1:250 did not work, did not see ANY bands except heavy & light chains ab414 1:250 did not work, did not see ANY bands except heavy & light chains WB attempt #2 ab412 1:100 no bands, except for heavy and light chains ab413 1:100 no bands, except for heavy and light chains ab414 1:100 no bands, except for heavy and light chains ADDITIONAL NOTES I am still trying to optimize my conditions and already I am running out of antibody. Will I continue to have to repeat these optimization conditions with every batch of ab412, 413, 414 I receive from your company? We know this protein is methylated, it is published data, which I have repeated several times in my lab. Background staining is not the problem, the problem is that your abs are not staining my protein of interest even though we know it is methylated.
Asked on Sep 30 2005
Thank you for your enquiry. Thank you also for completing our questionnaire so comprehensively. I am sorry that you have been having difficulties with our antisera against methyl arginine. I have examined your questionnaire and it seems as if you are following a protocol not dissimilar to Abcam's recommended protocol. You mentioned that your target protein is methylated. I have discovered that ICP27 is methylated at Arginine residues. I would have mentioned that your immunoprecipitation by H1119 may be obscured by the methylated form of the protein, resulting in the preferential precipitation of the unmethylated form. However, this does not explain the fact that you are NOT obtaining a result following western blotting using a crude HeLa extract. Given a crude extract the very least that I would expect to see would be the arginine methylated histones H3 and H4. In fact this is what I would recommend you use as positive control to guarantee that your abcam antisera are working and detecting methyl-arginine. Preparing a high concentrated histone extract can be done on HeLa cells. We have a very easy protocol at the link below. This will not affect your protocol given that you are using a 12.5% gel. Histones are between 10 and 17KDa. https://www.abcam.com/index.html?pageconfig=view_protocol&pid=316 You mention that you did have some success using a 1:250 dilution. Please can you e-mail me these scanned as JPeGs. I would also like to know the mass of protein you are loading onto the gel during the whole cell extract western blot and the relative amount you are loading following IP. I would recommend a starting amount of 20ug for the crude extract. Following some literature searching I would also like to know whether your ICP27 construct that you are injecting contains an RGG box as I have learnt that the methylation of ICP27 is dependent on its presence. I look forward to your reply and hope that we can resolve this issue quickly.
Answered on Oct 03 2005