western blotting problem: b-actin is uneven - (May/28/2006 )

I have measured the concentration of each sample and loaded the same amount of the total protein. I also tested it with Ponceau S, which shows even transfering and loading amount. However, the result turned out that some lanes have strong actin signal while some are very weak. I can't think of any possible reasons for this. Can you give me some suggestions? The more the better. Any possible and less possible causes of this?

Thanks a lot!

Hi
Check the answer I gave to mode vigilante in the recent topic entitled " protein lysate and blotting problems". Some of these considerations might suit your case as well.

Good luck

Thanks, but I don't think detergent is the problem. The beta-actin bands are very even, which can't be caused by unexact concentration determination. There must be some causes else.

OK. let's say the transfer is OK.
2 possibilities :
-actin expression is not even. Try an other marker
-you have a problem of detection. How did you do? ECL, with X-ray film exposure? could be due to air bubble between the membrane and the film.

Thanks!
I can't rule out the first possibility. I'll try tubulin next time. (I am using HEK293 cells)

Yes, I use Ecl but I don't use X-ray film any more, I use a machine to obtain the picture directly now. So, I don't think there are bubbles.

Hi. I'd prefer to look at actin signal and make normalization based on it instead of standardized protein conc. loaded. That's because if you're looking at protein expression, such as those induced or repressed, then the amount of proteins in EACH CELL will differ, so the most reliable marker here would be actin (assuming that actin is expressed equally in EACH cell, regardless of treatments or transfectiion) to ascertain that it represents CONTENT from EQUAL NO. of CELL loaded, instead of PROTEINS (which is variable, dependent on experimental treatments).

If you're using sample from different cell lines, assumption have to be made that each different cell line expresses actin (or tubulin or other housekeeping genes) ALMOST equally.

Anyway, just try using tubulin just to confirm and if the signal still differs, you can normalize your protein signal with signal from tubulin (or actin).

Dear Momuf,

can I ask what treatment you are doing to your cells before preparing the cell lysate?

Why: like all proteins, some treatments can actually affect the expression of house-keeping proteins. It is always good with a new treatment to test more than one house-keeping protein. As Isek suggested, maybe tubulin will work better for you although if the treatment is also affecting the actin-microtubule network, tubulin will not work either.

Another alternative is cyclophilin A and I can't remember the other right now.

Hope this helps,

AussieUSA.