4 replies to this topic

[RM82]

Hi all,

I have doing western blots for a while and am perplexed by some recent results.  I quantified all my protein samples by Bradford and loaded them on a gel.  When I blotted with tubulin (as loading control) I only saw a bands in 2 of my 5 samples.  The others had no bands.  I then decided to Ponceau stain my membrane and it showed equivalent loading of proteins in all 5 lanes!  I then tried blotting with a second loading control (actin) that other members of my lab use and had the same result...only 2 samples show protein.

What do you think I might be doing wrong? The ponceau clearly shows there is protein in all lanes and equivalent amounts yet when I tried actin and tubulin I see no bands in 3 of my lanes.  ps: this is not the first time this has happened!!!

[bob1]

How did you ponceau post blocking?  This should result in the bands being difficult to see as the block will bind to the membrane.

Perhaps your anibody incubations didn't work well - parts didn't get covered very well?

[RM82]

I usually don't Ponceau after blocking- but in this case I decided to try it to see if anything would show up and I saw clear bands (in the same pattern) in all the lanes.  There were a few blank lanes where I did not load any protein and these did not show any such bands upon Ponceau staining.

I'm starting to think it might be that some parts didn't get covered as well......its the only way I can explain this.  Any other suggestions?

[bob1]

I have found that with your sort of problem,it is usually due to the membrane not being covered in one of the solutions at some time.  This is especially the case if you use a bag to incubate the membrane - I find that 3-5 ml of each solution works well for bags. I haven't seen it happen so much with containers, but I would use a bigger volume there.

[RM82]

Thanks, thats my suspicion at this point as well. I'm running it again with more solution this time.