Question (17787) | Anti-RASSF1a antibody [3F3] (ab23950)

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Question

BATCH NUMBER 199599 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE the whole protein of human stomach tumor cells PRIMARY ANTIBODY ab23950, 1:100~1:1000, 4? overnight DETECTION METHOD ECLPlus POSITIVE AND NEGATIVE CONTROLS USED control: lung cells of human embryo ANTIBODY STORAGE CONDITIONS -20?, after aliquoting store at 4? SAMPLE PREPARATION lysis buffer:50mM Tris-Hcl,1mM EDTA,2%SDS,5mM DTT 10mM PMSF the cells were lysed 30min,then sonicated the lysates with ice. followed heated 10min and centrifugated to get the supernant. AMOUNT OF PROTEIN LOADED 100ug ELECTROPHORESIS/GEL CONDITIONS 10% PAGE, 80v 1h TRANSFER AND BLOCKING CONDITIONS 120v 2.5h, 5% or 1% fat free milk to block for 1.5 hour SECONDARY ANTIBODY BeiJing DingGuo CO.Lit. RT, 2 hours HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 8 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? 1?adjust the concentration of primary Ab, 1:1000,1:500,1:300,1:100 2?adjust blocking buffer from 5% fat free mlik to 1%.

Answer

Thank you for providing the customer's detailed protocol, it enabled me to investigate the problem rapidly and thoroughly. There are two main points I would like to concentrate on: 1) levels of the protein may be low in the customer's samples. Following some publication readings I found several references for low levels of RASSF1a in some gastric carcinomas. For example, Byun DS, Lee MG, Chae KS, Ryu BG, Chi SG.Frequent epigenetic inactivation of RASSF1A by aberrant promoter hypermethylation in human gastric adenocarcinoma.Cancer Res. 2001 Oct 1;61(19):7034-8. states "RASSF1A and RASSF1B transcripts were not expressed in 60% (9 of 15) and 33% (5 of 15) of gastric carcinoma cell lines". I think it is therefore important to run a positive control along the customer's samples. I would recommend to run a HeLa lysate. 2) the customer has no protease inhibitors in the lysis buffer, other than PMSF. This is not sufficient and therefore does not prevent enzymatic degradation of the protein. Please add a cocktail of inhibitors (sigma or Roche for example) to the buffer. The protein can be located in the nucleus so I recommend to use a stronger lysis buffer than a Tris buffer: please use a RIPA or NP40 buffer. (recipes for those buffers and detailed inhibitors can be found on our protocols page https://www.abcam.com/index.html?pageconfig=popular_protocols) Other small points to note are: - the customer mentions "after aliquoting store at 4C"; after aliquoting the antibody should be stored at -20C. Aliquots should be used straight after defrosting and discarded if not used but not kept at 4C for future uses. -the customer mentions lysates being heated for 10min. Can I please make sure this is in reducing loading buffer? 5 minutes at 95C is sufficient. Please make sure the gel is also reducing and check the transfer is adequate. -the customer does not mention what type of secondary is used. I expect it is an anti mouse HRP conjugated product. I recommend to check that it works well with other mouse primary antibodies. -a 12.5% gel would be more suitable than a 10% gel to resolve the protein better. -We find that blocking 1hr in BSA 5% can give better results than milk. Milk, if added in the antibody dilution buffer can also prevent the antibody from binding so if the customer adds milk to the antibody dilution buffer he should try without. Please make sure Tween 20 (0.1%) is present in the TBST antibody and wash buffers. I hope the above recommendations will resolve the customer's problems. Please let me know how he gets on and if he needs further assistance,

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