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        Troubleshooting Guide: Western Blot

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        Troubleshooting Guide: Western Blot


        Problem: Poor Transfer

        Possible Source Test or Action
        Membrane does not wet uniformly Pre-wet membrane in 100% methanol
        MW of protein is less than 10,000 If protein has MW <10,000, it may have �lown through?the membrane. Reduce transfer time, or place two sheets of transfer membrane soaked in Anode Buffer II under gel in transfer stack, or select membrane with smaller pore size
        Isoelectric point of protein is <9 Use alternate buffer system (such as CAPS buffer pH 10.5) with a higher pH

        Assemble transfer stack with additional membrane soaked in cathode buffer on the cathode side of gel. This will capture proteins that migrate towards the cathode. Identify membrane location in stack and immunostain all membranes in stack
        Methanol concentration Higher methanol concentration increases binding to membrane but may retard transfer from gel
        SDS concentration Adding 0.005 - 0.01% SDS to cathode buffer can increase transfer efficiency of protein from gel
        Note: Increased SDS concentration may interfere with binding to membrane
        Thick gel Thicker gels or higher MW proteins may require longer transfer times

        Problem: High Background Staining

        Possible Source Test or Action
        Membrane does not wet uniformly Pre-wet membrane in 100% methanol
        Inadequate blocking Optimize the blocking step by trying alternate blocking solutions (e.g. , non-fat milk, gelatin, etc.) or increasing time and temperature of blocking step
        Cross-reactivity of antibody reagents Check for cross-reactivity of antibody reagents to the blocking protein

        Problem: Low Protein Binding

        Possible Source Test or Action
        Overwashing Keep the length of TTBS washes to a minimum

        See �oor Transfer?problem for alternatives

        Problem: High Protein Binding, Low Signal

        Possible Source Test or Action
        Inadequate antibody staining Check antibody dilutions and expiration dates
        Inactive AP conjugate and/or substrate Add enzyme conjugate to substrate reagents as prepared in step 7 of immunostaining procedure. If color occurs, reagents are performing properly
        Poor sample If positive control worked, sample may not contain protein of interest or it may be present at concentrations too low to detect

        Problem: Several Bands are Stained

        Possible Source Test or Action
        Non-specific binding of secondary reagents Check non-specific binding by running extra sample lane and omitting 1?antibody from immunostaining procedure, run with a non-specific antibody control in place of 1?antibody, or perform dot blot and omit the 1?antibody. If staining occurs, choose an alternate 2?antibody
        Primary antibody is not monospecific Use a monoclonal or affinity purified antibody

         

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