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LOT NUMBER xx ORDER NUMBER xx DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE purified protein from sigma PRIMARY ANTIBODY Concentration or dilution 1/100 dilution (2.5ug/ml) Diluent buffer 0.5% BSA in PBST Incubation time overnight (16hrs) Incubation temperature: 4 degree C wash buffer PBST Number of washes 10min RT, 3 times DETECTION METHOD LAS4000 chemiluminescence POSITIVE AND NEGATIVE CONTROLS USED Positive control Beta-casein (Sigma, C6905) Negative control not used ANTIBODY STORAGE CONDITIONS -20 degree C SAMPLE PREPARATION Lysis buffer : not used (resuspension buffer : 20mM Tris-Cl pH 8.0) Protease inhibitors: not used Phosphatase inhibitors : not used Reducing agent DTT Boiling for 5 min? yes/no yes AMOUNT OF PROTEIN LOADED 1ug/lane ELECTROPHORESIS/GEL CONDITIONS 10% Tris-glycine TRANSFER AND BLOCKING CONDITIONS Type of membrane PVDF Protein transfer verified yes (with prestained marker) Blocking agent and concentration 3% BSA, 0.1% gelatin in PBST Blocking time 2hrs Blocking temperature RT SECONDARY ANTIBODY not used HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A NO PRIMARY CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? - ADDITIONAL NOTES -
Asked on Feb 13 2012
Thank you for taking time to liaise with the customer and to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.
The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.
I have reviewed this case and would like to confirm the following points with the customer:
1.) In the image provided, the band of the casein protein seems to have a strange migration. Has the customer tested this casein protein on another gel with the same results?
2.)As general suggestion, I can advise to keep the samples all the time on ice for detection of phosphoprotein. I do also recommend the use of phosphatase inhibitors. As I suppose however that the MS data and the gel have been performed with the same sample of casein, I do acknowledge that the casein protein has been phosphorylated and that therefore the positive control should provide a signal. See also our protocol for phosphoproteins. https://www.abcam.com/ps/pdf/protocols/Western%20blotting%20of%20phospho-proteins%20protocol.pdf
In the event thatthis antibodyhas not worked on different blots detecting the phosphorylated casein, we would be pleased to provide a free of charge replacement orcredit note.
I am looking forward to hear back from you.
Answered on Feb 13 2012