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many bands Western Blot


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#1 fabipp

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Posted 04 April 2012 - 03:11 AM

I am having a trouble with my western blot analysis. I am working with a 90Kda, but because it is a tagged protein, the weight increase 20Kda. It is a protein TAP tagged C. albicans and to detetct it I use TAP antibody (Anti Tap Polyclonal Antibody) from Thermo Scientific. The second antibody is against rabbit. I am using a dilution of the 1st ab: 1:10.000 and the second 1:5000. I can see a lot of bands with the same intensity. i am sending you the picture, that you can see better my problem. In the picture I made a titulation of the 1st antibody. From the left side to the right side: 1:2.500; 1:5000 and 1:10.000

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Edited by fabipp, 04 April 2012 - 03:13 AM.


#2 Inmost sun

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Posted 04 April 2012 - 03:16 AM

do you have a control peptide carrying TAP to run negative IB controls? extra bands could be specific (recombinants or splicing forms each fused with TAP) or unspecific bindings which may be reduced by lower titer of primary antibody...

#3 fabipp

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Posted 04 April 2012 - 03:22 AM

No, I do not. But I wiil try to get it. Thanks




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