Huge background in Western blot - Studying the histone modification (May/09/2011 )
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
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.