Blotchy/White Spotted Membranes

[littad]

has anyone experienced blotchy/white spotted membranes before? the blotchiness affects the band intensity and causes erroneous quantitation.

here's a brief overview of my protocol from the start of the transfer.

1. activate PVDF membrane in MeOH
2. equilibrate PVDF membrane in transfer buffer (1X NuPAGE Transfer Buffer, 20% MeOH) for 1-2 hours.
3. transfer for 3 hours at 100V at 4C
4. block membranes in 5% milk in TBST 0.1% for 1-1.5 hours
5. wash 2-3 times in 10 minute intervals in TBST 0.1%
6. add primary overnight at 4C
7. wash 5-6 times in 5-10 minute intervals in TBST 0.1%
8. add secondary in a 1:10000 dilution in 5% milk in TBST 0.1%; rock at room temperature for 1 hour
9. wash every 10-15 minutes for 2 hours in TBST 0.1%
10. develop with amersham ECL+ kit

could bacterial contamination cause the blotchiness?

[dnamolecule]

Hello there,
welcome to the forums.

You must have had bubbles on transferring to your membrane.

If its lighter/white coloured on the x-ray film then it has to be bad transferring , possibly incubation problems (with your antibody).

Could you post your gel for us to see?

cheers

[littad]

Originally Posted by dnamolecule

Hello there,
welcome to the forums.

You must have had bubbles on transferring to your membrane.

If its lighter/white coloured on the x-ray film then it has to be bad transferring , possibly incubation problems (with your antibody).

Could you post your gel for us to see?

cheers

I don't think it's the transfer step... the reason being is that the splotchy/blotchy patternation is different after reprobing. The film is below (attached to the post), thanks!