TROUBLESHOOTING GUIDE
Contents
1. e immunocomplex Too many proteins in mixture e Centrifuge lysate at 100 000 x g for another 30 minutes to remove any extra fragments Protein only available in low levels Increase antibody concentration in the sample type e Increase cell lysate concentration e Metabolically label cellular proteins StressMarq Biosciences Inc 1 250 294 906 info stressmarq com Possible Cause Possible Solutions Specific Other Interfering substances e Be cognizant of pH excessive detergent Antigen Not concentrations and reducing agents such as Detected DTT and _ mercaptoethanol Cont Incorrect bead type used e Make sure the right ones were used and re try Immune complex was stripped from Use a milder wash buffer agarose beads by wash buffer e Change detergents to something with less salt and or a lower detergent concentration e Reduce number of washes can sit overnight at 4 C prepared yourself StressMarq Biosciences Inc 1 250 294 906 info stressmarq com2. efoto StressMarq BIOSCIENCES we TROUBLESHOOTING GUIDE IMMUNOPRECIPITATION Possible Cause Possible Solutions Non Specific Insufficient washing Use more stringent washes Background e Alternate wash buffers from high to low salt e Use a different detergent e Have one wash be with distilled water Increase the number and time of washes Still frozen lysates e Do not freeze before use High antibody concentrations Non specific binding to agarose e Pre clear lysates beads or antibodies Non specific binding to Proteins e Preload precipitated antibody then block remaining sites with BSA gelatin acetone powders or 5 nonfat dry milk Aggregated proteins in lysate e Prior to adding the antibody thoroughly centrifuge at 100 000 x g for 30 minutes immunoprecipitation Specific Polyclonal antiserum protein Background complexes formed polyclonal Antigen consists of more than one polypeptide chain than one polypeptide chain Monoclonal or affinity purified e Use a monoclonal with a different epitope polyclonal antibody recognizing homologous epitope Immunoblots are actually Ig light or IgG light chains are recognized at 28kDa heavy chains IgG heavy chains are recognized at 55kDa Specific Non suitable antibody e Try a different antibody Sometimes Antigen Not polyclonals work better Detected Antibody concentration too low e Increase the concentration Weak binding to the proteins e Use a bridging antibody to captur
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