Troubleshooting the Western blot procedure
Contents
1. Analysis Procedures Western blot Troubleshooting the Western blot procedure Problem Chemiluminescent or chromogenic signal weak or not visible Possible cause Remedy Poor Isolation of tagged protein Use a different cell lysis procedure Antibody too dilute Double the concentration of the Tag specific Antibody and or the Secondary Antibody Too little protein on the gel Add more protein to gel Poor transfer of proteins from gel to membrane Note Check efficiency of transfer by silver staining the remaining gel There should be minimal protein left in the gel 1 Increase the electrical current and or the transfer time for the blot 2 Be sure there are no air bubbles between the membrane and gel during transfer Wrong type membrane For maximum signal use PVDF membranes for transfer Antibody incubation too short Incubate the Tag specific Antibody and or the Secondary Antibody with the membrane blot for a longer time Signal development time too short Double the development time Wash time too long or too stringent 1 Shorten the washing time 2 Omit Tween 20 from the Wash Buffer Enzyme on antibody conjugate inactivated by preservative Note To check enzyme activity dot increasing dilutions of the antibody conjugate onto a membrane and perform the visualization reaction directly on the dilutions If you use POD conjugated antibodies do not use so2. 5 w v dissolved in Reagent Diluent as Blocking Reagent Caution High concentrations of nonfat dry milk may reduce specific signal as well as background Contaminated reagents or equipment 1 Use clean equipment freshly prepared buffers and new membranes 2 Always avoid touching membranes with bare hands use gloves and forceps Secondary antibody binds untagged proteins Use an F ab fragment of a secondary antibody rather than an intact IgG Heavy and light chains of primary antibody visible on blot membrane Indirect detection procedures only Use direct detection with peroxidase conjugated monoclonal antibody to visualize tagged proteins Signal development time too long Reduce development time by half lt q CONTENTS
3. dium azide in any Western blot reagent If you use AP conjugated antibodies do not use a phosphate buffer in the washes following the incubation with Secondary Antibody substitute a Tris buffer Substrate inactive Note Check reagent on dot blot containing dilutions of antibody conjugate with established enzyme activity Make fresh dilution of substrate or start with a different stock of substrate Epitope tag sequence is not detectable due to e Proteolytic cleavage e Low level of expression Premature translation termination resulting in loss of C terminal tag sequence Include protease inhibitors in lysis buffer Use alternative expression system or optimize your expression system Insert multiple tag sequences into target protein to increase avidity of antibody reaction Use alternative insertion site within the target gene for the epitope tag sequence lt q CONTENTS Analysis Procedures Western blot Problem High background additional bands on blot Possible cause Remedy Antibody too concentrated Decrease concentration of Tag specific Antibody and or Secondary Antibody by half Wash time too short Increase washing times Incubation of membrane with substrate too long Leave blot membrane in substrate for a shorter time Wrong type membrane For minimum background use PVDF mem branes for transfer Blocking Reagent too dilute Use nonfat dry milk
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