Hello,
I am attempting to image my membrane with an ECL solution but I keep getting this mess as a result. I'm not sure where the error could have been made (blocking, primary incubation). I ran my gel at 25 mA, transferred at 55V for 2h, blocked for 1 hour (5% milk), then incubated with primary (conjugated with HRP) overnight. Has anyone seen something like this before during imaging? Could this be a wash problem? Thank you!
Hi Anthony Bishay
This blot is of your protein of interest or housekeeping protein? How was your housekeeping protein result? Do this 2hr transfer at 25mA is standardized by you?
Activate the PVDF membrane for at least 1min in methanol. Try with fresh primary and secondary antibodies, also keep any positive samples for the respective antibody.
Which company transfer machine it is?
Regards
Saurabh
Hi! Anthony,
As Saurabh pointed out, positive control is essential at this stage. Judging from the molecular weight marker patterns on the left sidelines, it is possible that the transfer (blotting) is not performed accurately (such as uneven adhesion between the gel and the membrane).
Shintaro
Hi Anthony! as both Saurabh and Shinatro pointed out, it looks like a problem related to the transfer. Are you using PVDF membrane? If so, you need to incubate it in Methanol (1-5 min) before use and then equilibrate it in transfer buffer for 5-10 min, before preparing the blotting sandwich. Let us know how you solved this issue and good luck!
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