Hi everyone,
We have faced some problems with our new western blot experiment.
Some columns have high background and in others/same column some black spots appear.
Coincidentally or not, when we perform the same western blotting, with same samples, appear black spots in the same columns, but not with the same pattern.
We worked with tick organ extract as antigen and bovine serum as antibody.
As washing solution we use PBS + Tween 20.
As blocking solution we use PBS + Tween 20 + fat-free milk.
Our conjugated antibody has high quality (Sigma), as well as our western blotting substrate (Pierce ECL Plus – ThermoFisher Scientific).
We believe the high background in some samples even can be related with the serum quality, but we can not explain or even solve the black spots problem.
Could somebody help us?
For the background problems that I had, I only had to change the dilution of my 2nd antibody to a lower concentration (or higher dilution), or sometimes the concentration of both, 1st and 2nd. So then, when I revealed the membrane it looked cleaner.
Here are my suggestions:
1) higher concentration of primary and/or specially secondary antibody: This could be the main reason. Decrease antibody concentration
2) Lack of proper washing after antibody incubation: try to wash it longer
3) Skim milk is not dissolved properly: dissolve skim milk well or filter it after dissolution
Good luck.
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