Dirty blots/high background - (Mar/22/2016 )

Hi,

I am having trouble with a very high background on my blots (image 1). I have run 2D gels and then transferred. I block with 5% skim milk/PBST and primary and secondary antibodies are also in 5% skim milk/PBST.

I have been using this technique for some time and have up until now had rather nice clean blots (third image).

The difference with this batch is that the protein on the 2D gel is different, although the 2D gels are good so it's not a problem with that (image 2). The secondary antibody was from a fresh batch this time.

any thoughts on this? Is it too high concentration of secondary antibody?

is your primary against phosphoproteins?

if so then you may want to try a different blocking agent.

Hi,

No, the primary is patient serum. We're probing for autoantibodies in sera of patients with disease. I just don't understand how one blot can be fine and then the next series of blots are so dirty

thanks

do you use fresh solutions for each blot or do you reuse some (eg block, secondary antibody).

if so then the blocking agent may be depleted.

do you ensure that conditions during processing are maintained run to run?

Make sure you adjust the iris in the imager machine properly, I often encountered this when I observed my western blot in chemidoc. The previous user set it way too high, back then I wasn't aware the iris should be as low as possible (yet still manage to see clearly), and so the image look like dirty blots and I assumed as high background. After numerous painful repetition, only I realised what was the problem. 

How about your sample concentration? Do you reuse your primary antibody or second antibody? And how about the dilution ratio about the antibody, dose the dilution ratio suitable?