2D PAGE and Western blotting - (Jan/19/2016 )

Hi,

Just looking some input on this, as an outsider's opinion is always appreciated!

I'm looking for novel auto-antibodies in patients with disease versus healthy controls. The experiment setup is as follows:

I am extracting protein from a human cell line (specific to the disease) and running 2D PAGE and then transferring the gel by Western blot. The Western blot is then probed with the patients plasma to see if anything binds. I have 30 patients in my disease group and 30 in my control group.

So, in theory I will be running 60 gels/western blots.

The problem arises when I come to compare them. How would I even go about this? I would need to compare within each group and then compare bewteen groups. I have Progenesis software.

Any input would be great including any issues that are obvious?

many thanks

Controls, controls, controls. Controls are a biologist's best friend.

You want to control for total protein difference, so that the difference in disease protein signal is normalized. I'm not an expert in this, but I think you could use a housekeeping gene as a control. You could multiplex your Western with an antibody against your disease protein and one against a housekeeping protein. They use housekeeping genes in a similar way in qPCR.

60 Westerns is like... several months of work. You should look for an easier way, something high-throughput.

What do you mean when you say you're probing with plasma? What is your secondary antibody, what does it bind to?

For between groups, I'm guessing you could take the mean of each group and do a t-test. Or else, maybe an ANOVA? I'm not an expert on statistics but that sounds like it could help you.

Within groups should be easy; ANOVE to compare all at the same time, or paired t-tests (would be a LOT) for the disease protein.

I am wondering like... if there is some way to pool your samples and then isotope label so you can do mass spec. That might eliminate individual patient data though, only allow for comparison between groups. But that's one half of your question, so.

Curious to see what you end up doing!