I am getting a high background problem with western blot. I have given long time for blocking and washing with 1% TBST thrice for 10 minutes. But the result does not improve and the background is higher than our bands while beta actin is OK. Could anyone help me out this problem?
Did you use primary antibody in blocking buffer? What concentration are you using?
What is your blocking solution? Is your secondary antibody the same as for detecting actin?
Yep I used the primary Ab in 5% milk in TBST. I am using 1:1000 dilution of primary Ab. My blocking solution is 5% milk which we are using commonly in our lab. The secondary Ab is the same for both.
Okay. So to begin with how about you use 1:1500 to 1:2000 for primary antibody? can you post your blot scan as well.
You can try using BSA instead of 5% milk- also add the secondary Ab only for 1 hr at RT.
Yes Zaid I am using the secondary Ab for 1 hr at room temperature. May be BSA would work instead of 5% milk.
In addition to dilutions and BSA you could add 5 to 10% of serum of the animal your secondary Ab were produced. It helps a lot.
Thanks Monica. I hope so the combination would be better than alone. I will try it.
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