philman

yeah the band in the image is supposed to be B actin, and it is supposed to be in all 7 wells not just one of them! The only other antibodies on the membrane were for P-ATF2 which is around 70kD, sorry I didn't mark the band sizes! I didn't use coomassie, but I use Ponceau staining quite alot, as it is reversible  and with Ponceau I see lots of bands, of relitavely equal staining.

The rest of my lab hasn't wanted to try my secondary now that I am having problems! But someone is trying it out this afternoon with their loading control so we'll see if it works then.

No one else is looking for the ATF2 as I am so they haven't been using my antibody, but I could ask one of them to give it a go at some point. And the buffers aren't ubiquitously used, but we all use the same stock chemicals and recipes to make up our own buffers.

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Erm, so your running buffer is at 2x concentration? You should only need to add 100ml 10X buffer to a 1L solution.

And I haven't heard of adding methanol to the running buffer, to the transfer buffer, sure, that is common, but not to the running buffer. Have you mixed up your protocols perhaps?

I was just asking about the conjugated antibody controls as we already have those and they are not being used, so it would save spending another £100+ on more non-conjugated controls.

I will take your advice bob1 and try the clearing with just the beads then, and hope that is enough.

Thanks!

I have never had much of a problem with milk background staining, and there isn't much background on the blot, so I don't think that is a problem, although I will block with BSA next time to see if it makes a difference.

I have attatched the image with the faint bands, you can see there is little background. This is after 20 minutes exposure!

 25th june P-Tyr.jpg   46.95K   158 downloads