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If your product does not perform as described on this datasheet, we will refund or replace your product...
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BATCH NUMBER 87396 ORDER NUMBER 1003198 DESCRIPTION OF THE PROBLEM No bands SAMPLE Human Endometrial Stromal Cells PRIMARY ANTIBODY Abcam - Mouse monoclonal [1.B.78] to PRG2 1 in 100 dilution Incubated overnight at 4 degrees Wash for 15 minutes DETECTION METHOD ECLPlus (Supersignal WestPico Chemiluminescent Substrate (Pierce) POSITIVE AND NEGATIVE CONTROLS USED None - can you recommend one? ANTIBODY STORAGE CONDITIONS 4 degrees SAMPLE PREPARATION RIPA lysis buffer (Upstate cell signalling solutions) Complete MINI Protease inhibitor (Roche diagnostics) AMOUNT OF PROTEIN LOADED 20 micrograms ELECTROPHORESIS/GEL CONDITIONS 4-15% gradient gel (BioRad ready gels 4-15% Tris-HCl) Reducing gel TRANSFER AND BLOCKING CONDITIONS Transfer for 30 mins at 15 volts. Buffer = Western Transfer Buffer (25mM Tris (3g/L), 0.2M glycine (15g/L), 20% methanol) Block in 5% milk solution for 30 minutes SECONDARY ANTIBODY Sheep Anti-Mouse IgG HRP conjugate [another supplier] 1 in 2500 Incubated for 1 hour at room temperature in 5% milk solution Wash for 40 minutes 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? Added more protein (originally added 10 micrograms) |
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ANSWER: |
I have received confirmation from the laboratory that tested ab14462 that your protocol is very good and should work with ab14462. The laboratory shared also the following feedback which may interest you "Your customer's secondary antibody is a Sheep x mouse IgG HRP. Ab14462 is a mouse IgG1. I have had diverse experiences with this sort of distinction. I have had customers state that an anti-IgG will recognize all IgG's of the same species; and yet there have been times when I suggested matching the secondary precisely to the target isotype and suddenly the protocol was working." As the antibody did not work for you I would be happy to offer you a replacement vial or credit note/refund. Please let me know which you would prefer and I will arrange this, I look forward to hearing from you,
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I have been using PRG2 antibody [1.B.787] in Western Blots, but have not had any success. Could you recommend a concentration of this antibody to use for direct ELISA? |
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ANSWER: |
I have been informed by the source of ab14462 that it was not tested in house in ELISA and that unfortunately we therefore do not know the recommended dilution. I was recommended to start at a concentration of 2-5ug/ml (for Direct or Indirect ELISA), incubating overnight at 4C. I'm sorry I cannot provide further help. I will look into your complaint as soon as possible and appreciate your patience, |
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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"