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DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE HuCCT1, PanC1 whole cell lysates PRIMARY ANTIBODY Concentration or dilution : 1:1,000 Diluent buffer : 1% skim milk in PBST Incubation time : O/N Incubation temperature: at 4℃ wash Buffer : PBST Number of washes : 6 times(each for 5 min) DETECTION METHOD LAS-3000 POSITIVE AND NEGATIVE CONTROLS USED Positive control : PanC1 p8 has been shown to be highly expressed in PanC1 cells (PMID: 21344397) ANTIBODY STORAGE CONDITIONS Store at -20℃ SAMPLE PREPARATION Lysis buffer : RIPA buffer Protease inhibitors: 2mM PMSF and 1x PIC Phosphatase inhibitors : no Reducing agent : yes Boiling for ≥5 min? yes. AMOUNT OF PROTEIN LOADED Protein loaded ug : 50ug protein ELECTROPHORESIS/GEL CONDITIONS 20% SDS-PAGE gel TRANSFER AND BLOCKING CONDITIONS Type of membrane : PVDF Protein transfer verified : protein transfer was verified by dye transfer and commassie stainingof the gel Blocking agent and concentration : 1% skim milk in PBST Blocking time : 1 hour Blocking temperature : Room temperature SECONDARY ANTIBODY Jackson ImmunoResearch anti-Rabbit igG-HRP Concentration or dilution : 1:10,000 Diluent buffer : in PBST Incubation time : 2 hours Incubation temperature: room temperature wash Buffer : PBST Number of washes : 6 times(each for 5 min) 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? Blocking : 1% skim or 2% BSA Transfer : 1 hr or 40 min Primary Ab incubation : O/N or 3days ADDITIONAL NOTES Its predicted size is 8kDa but, some references showed 15~29kDa bands. There are non-specific bands only and any of them showed knock out pattern with siRNA treated sample.
Asked on Sep 21 2011
Thank you for taking time 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. Having reviewed this case, it appears that ab6028 is not performing as well as might be expected. I would therefore like to offer you a free of charge replacement of this antibody with another from our catalogue. We have 4 other primary antibodies directed against p8: ab46889, ab93964, ab94034 and ab87454. All of which are suitable for WB. Please note ab87454 is a mouse monoclonal. Please let me know which antibody you would like to try and I will arrange it for you. Additionally, some suggestions which may help to optimise the results from ab6028 and the antibody which you choose to replace it with. -We would typically recommend blocking using 5% BSA solution -As the protein is small I would suggest removing the SDS from the transfer buffer if you haven't already as this hinders the binding to the membrane. I would also suggest including 20% methanol in the transfer buffer. -You may want to try reducing the transfer time as the protein is small it may migrate through the membrane too fast to be adsorbed efficiently More useful information may be found here: https://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund. I hope this information is helpful, if you have any further questions please don't hesitate to ask.
Answered on Sep 21 2011