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【分享】Western blot 常见问题及解决方案
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转自http://www.molecularstation.com/ ... western-blots/#weak Troubleshooting Western Blots Problem: Spots on Film (missing bands) : - Poor Transfer due to air bubbles during transfer to membrane - dirty film when adding to the developer - developer rolls are dirty Solution to spots on film: - use a pasteur pipette as a roller. roll the membrane and gel before transfer to remove bubbles. keep membrane and materials wet. - keep film clean before adding to the developer Troubleshooting Western Blots Problem: Too Many Bands in Western Blot : - usually due to many non-specific bands - poor primary antibody quality or old antibody - not enough blocking - proteolytic cleavage of antigen - all additional bands are lower MW than your protein, ( ie your protein is being detected but was degraded) Solutions to too many bands on western blot: - purchase another antibody or replace with a fresh antibody stock - block with 5% dry non-fat milk or BSA prior to adding primary. If you are already blocking, consider blocking during primary and secondary antibody incubations and also increasing blocking milk or BSA concentration. - keep everything on ice, use fresh samples, store in - 80C, and use protease inhibitors such as PMSF Troubleshooting Western Blots Problem: High background; low signal to noise ratio on Western Blot - black or every dark film; film was exposed too long - blocking was insufficient ; non-specific binding of primary antibody and secondary antibodies were not washed completely - washing was insufficient - film glows in dark! - too high secondary or primary dilutions Solutions to Dark Film: - expose for less time, decrease exposure time - increase blocking duration of non-fat dry milk 5% incubation or increase the concentration. - also you can try blocking with whole serum of the host animal with (or before) the secondary antibody - increase washing times and volumes to help remove any non-specific signal due to weak antibody binding. - using a stronger detergent to wash - Instead of TBST (Tween-20), use stronger detergents such as NP-40 or SDS which may provide a more stringent wash and reduce background (do this only if you band is strong! - washing also removes some your band of interest!) - very high secondary antibody (or even primary) concentration - perform optimization experiments to determine proper antibody dilutions. dilute the antibodies further. Troubleshooting Western Blots Problem: Low Signal or Weak Signal - due to not enough protein loaded on the gel - primary antibody is old or not good - phospho-proteins usually need overnight primary antibody incubation - not enough secondary antibody - over-blocking - developing reagents are bad / you made a mistake when preparing them Solutions to Low Signal or Weak Signal: - load more protein sample onto gel - try fresh primary antibody - incubate primary antibody overnite for phosphoproteins at 4 degrees C (cold room shaker) - increase concentration of protein (lyse samples in less lysis buffer) - decrease concentration of blocking agent - check the developing reagents; prepare fresh ECL and re-ECL the membrane - increase the concentration or the length of primary antibody incubation period - increase concentration or the length of secondary antibody incubation period Fuzzy Bands, Bands Smeared, Bands not Sharp - bands smeared due to hot gel - bands fuzzy due to high voltage Solutions to Fuzzy bands and Un-sharp Bands: - decrease voltage or current, run in cold room, and prepare new running buffer. ( heat causes the gel to lose its rigidity and its resolving power) - pre-soak transfer membrane in the appropriate transfer solution (determ |
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