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Western Blotting


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#1 saeko

saeko

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Posted 18 June 2002 - 09:08 PM

Hi everyone.

I have been having these problems in Western Blotting.

I loaded only sample buffer or other proteins which do not include the protein of my interest(lysozyme), then after color development I see a band at the position of lysozyme in those lanes.  
I did dot-blotting of these samples without lysozyme, then they did not show the presence of lysozyme there.
I tried two different lysozyme polyclonal antibodies, and both showed those fake bands in the membrane after blotting of proteins from the gel but not in dot-blotting.
I diluted the antibody, then I did not see those fake bands, but the sensitivity to lysozyme was lowered.

Another problem is the cross reactivity of antibody to proteins in MW markers.
The whole lane of markers(I tried LMW and BMW from Bio-rad) was steined and each protein bands were clearly appeared.
Why do the antibodies (I tried 2 different antibodies) react with the proteins in the markers?

I am really confused (especially by the fake bands!), so if anyone has any advice or suggestion, please reply here.

Thanks.





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