You did not write how you are blocking the membrane and what membrane you are using. I use 2% BSA in H20 as a blocking agent and it worked well for me. Also titrate the antibody dilution. Add tween20 to 0.1% in the washing buffer.
Hi there,
The high background is due either to unefficient blocking or/and to low dilution factor for antibody. Usually 3% non fat milk in TBST is OK for blocking and antibody dilutions and TBST (0.1% Tween20) is OK for all washes steps and test higher dilution factors for primary antibody.
Mr. Verma,
Try to change the blocking solution. If you are using BSA, try either skimmed milk or roti-block. Use the same concentration of blocking solution for primary dilution as well. (if the background is arising from lanes means the sample/ Antibody might considered for trouble shooting)
Hi Mr. Verma,
Yeah, as people have mentioned above, you sound like you need to up the concentration of your blocking buffer (as high as 5-10% skim milk has worked well for us) for about 2 hours at room temperature. You can also try diluting your antibodies in a skim (or BSA) solution and playing around with the concentration.
@Geoff Margison sorry for delay inreply.. this type of image I m getting after several washing and changing duration time of incubation for 1AB and 2AB ALSO
It it due to overexposure of the x-ray film. Also you can further dilute your antibodies
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