The results of western blotting always show some non-specific blots, I wonder why it could happen, how to define the non-specific blots and how I can explain these blots when analyzing WB results.
Non specific blots may be related to the usage of the polyclonal antibody used to detection in the process of Immunoblotting. Polyclonal antibody is able to detect various antigenic structure, in comparison to the monoclonal antibody which is much more specific in the detection.
But when use monoclonal antibody, non-specific blots sometime still exist. Why this would happen?
what I did was I made primary antibody solution in 1X TBS with 0.1% tween-20. This might work .
The detection of antibodies depends on which antigen epitopes that the antibody recognises. You may want to increase the washing steps and the blocking duration to reduce non specific binding. We include milk or casein in the washing and blocking solutions
The non specificity may be avoided by washing 6 times for 10 mins each in enough volume of TBS-T (0.1%) and using lower dilution of Primary and secondary antibody, shorter exposure time to the membrane. You have to twik between the different steps you can considerably reduce the non-specificity. Sometimes changing the company of the Antibody also affect this thing!!! All the best!
Thank you for your kind respond, maybe the problem was caused by different company of the antibody. So I decide to change another antibody and recreate my experiment.
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