Question (20437) | Anti-NFATC4 antibody (ab3447)

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Question

BATCH NUMBER 306063 ORDER NUMBER 230325 DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE human B cell lymphoma cell line whole cell extract PRIMARY ANTIBODY Abcam anti-NFATc4 was used Recommended dilution 1:10 000, as well as 1:5000 used Antibody was diluted in TBST with 1% BSA, and membrane was incubated at 4 deg celcius overnight Membrane was then washed with TBST thrice, 10 min each time on shaker DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED Bands obtained using same sample with Santa Cruz's anti-NFATc4 ANTIBODY STORAGE CONDITIONS Antibody was aliquoted and stored at -30 deg celcius upon arrival SAMPLE PREPARATION Proteins were extracted using RIPA buffer with protease inhibitors. Protein was heated with 4x loading dye at 95 deg celcius for 5 mins, before being loaded. AMOUNT OF PROTEIN LOADED 40ug ELECTROPHORESIS/GEL CONDITIONS 6% SDS-PAGE gel used. 80V was used initially when proteins were migrating through the stacking gel, and increased to 140V thereafter. TRANSFER AND BLOCKING CONDITIONS Transfer to PVDF memebrane was carried out at 4 deg celcius overnight at 30V. Transfer buffer composition [2.9g glycine, 5.8g Tris, 0.37g SDS, pH8.3] Membrane was blocked in 5% BSA for 1 h at room temperature. SECONDARY ANTIBODY Santa Cruz Biotechnology goat-anti-rabbit IgG-HRP used Dilution 1:1000 in TBST with 1% BSA Membrane was incubated for 1 h at room temperature, and subsequently washed thrice in TBST, 10 min per wash with agitation HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? As recommended in answer for previous enquiry CCE1098662 - using a human protein sample and replacing the milk with BSA

Answer

Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab3447 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered. I have looked through the protocols you used and would like to confirm the following items. - can you confirm that the second antibody you used also managed to produce good results with other primary antibodies? In some cases, the problem may be with the secondary antibody not working. - could the no signal you are experiencing be due to poor transfer of protein to membrane? Did you check the transfer efficiency? This can be simply done with a reversible stain such as Ponceau S. I hope the above recommendations may already help you. If you have already tried the above suggestions and still experience problems, I will be more than happy to offer you a replacement or refund. In which case, please confirm your shipping address/purchasing agent information. Also, please advice me on how you would like to proceed with your enquiry, so that I can immediately arrange for a replacement or refund for you.

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