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BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 121940 DESCRIPTION OF THE PROBLEM In the description of the biotinylated antibody against SLPI it states that the antibody does not recognize cleaved portions or domains of the protein, only the intact protein. I have found that this particular antibody recognizes both intact and cleaved protein under reducing and non-reducing conditions the same as the non-biotinylated antibody. I would love to find out how you were able to get the antibody to react differently to the two different species of protein as I would like to design an assay around that functionality. SAMPLE Purified SLPI protein (recombinant) from R&D Systems. PRIMARY ANTIBODY anti-SLPI biotin or anti-SLPI purified (Abcam ) DETECTION METHOD ECL (Pierce) POSITIVE AND NEGATIVE CONTROLS USED Undigested protein, no protein, markers, ANTIBODY STORAGE CONDITIONS 4oC refrigerator in the dark SAMPLE PREPARATION 1mg/ml in 1X PBS AMOUNT OF PROTEIN LOADED 20ug ELECTROPHORESIS/GEL CONDITIONS Many conditions, all show same result. TRANSFER AND BLOCKING CONDITIONS Invitrogen NuPAGE system SECONDARY ANTIBODY anti-mouse=HRP, or Streptavidin=HRP HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Reducing vs. Non-reducing BME vs. DTT Tris-glycine/MES vs. TRICINE ADDITIONAL NOTES Good luck, we're all counting on you...
Asked on Feb 08 2006
We forwarded your question to an expert in this field and this is the answer I received: The reactivity of the antibody with the isolated domains was evaluated using isolated domains of SLPI obtained by cleavage using partial acidic hydrolysis. Therefore, it is entirely possible that the antibody does recognize other SLPI cleavage products generated by e.g. (microbial) proteases. I have removed the sentence on the datasheet which originated from a customer feedback and hope this information helps you understand your results. I'm very sorry this sentence was not correct.
Answered on Feb 17 2006