The background of my Western Blots is very uneven. I use premade NUPAGE gels and PVDF membrane. For blocking I using intercept blocking buffer for 1 hour at room temperature. Primary antibody is incubated overnight and secondary for 1 hour at room temperature. I keep the membrane in a 50 ml tube and it is on a rollerbank during all the incubations to keep it from drying out. Does anyone know what causes the background on the blots and how I can fix it?
Thanks in advance!!
My guess, the 50ml tube is the culprit. Your membrane is to big to fit in streight so you have to role it, right? This way you have multiple layers of membrane and diffusion of your blocking agent might be poor. Use a simple flat dis instead.
Option B, did you check your gel by coomasie staining? There might be something with the gel running. Also, how did the markers look?
is it wrapped in clingfilm?
If it is wet it with methanol, this stops any static occurring between the clingfilm.
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