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Thread: western blot

  1. #1 western blot 
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    What specific steps of wetern blot can modify to improve specificity?


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  3. #2  
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    Is this a homework question?


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  4. #3  
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    I'm no specialist... exchange antibodies (in particular the first antibody), different membrane, gel with smaller mash openings e.g. by increasing the concentration of Acrylamide.
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  5. #4  
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    Hello there!,

    You can do the following to improve specificty:

    Lower protein loading or concentration in gel,

    Blocking membranes longer or using stronger blocking buffer (10% milk usually),

    Use lower antibody concentration,

    Use more specific antibody (i.e. better antibody

    Remember better specificty usually means lower sensitivity.
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  6. #5  
    Forum Masters Degree samcdkey's Avatar
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    In addition to what Western Blot guy has said, I would also recommend better washing and some experimentation with the levels of TBST [I used to optimise by first standardising the protein load and antibody concentration, then the time and concentration of blocking and finally the levels of TBST in the washing buffer]
    I also find that low concentrations of secondary antibody make for less background and cleaner blots.
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