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Huge background in Western blot

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#1 scifistudent



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Posted 09 May 2011 - 02:51 AM

Hi all,

Here i want your worthy opinion on my blot attached herewith. I probed with total histone (1:20K, 2h).Concentration of the protein loaded on the gel is 15 ug per well. I developed the blot and found a huge background. Can anybody suggest me the neccesary step i should adopt to study the total histone (17kd).

thanx for futher advice
sikander histone.jpg

#2 mdfenko


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Posted 09 May 2011 - 08:13 AM

tell us more. what is your procedure and solutions?

from what i can see, your problem is not background but may be lack of specificity (too many bands). that can be corrected by diluting the antibody and/or adding tween-20, up to 0.1%, to your buffers.

you should determine if the cause is the primary or secondary antibody.

Edited by mdfenko, 09 May 2011 - 08:18 AM.

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