I was wondering about same problem if anybody can help me, I will appreciate for that. Thanks...

One explanation would be degradation of some proteins in the lysate, including f-actin in your case,  or damage also by degradation of the epitope targeted by the antibody. In both case the problem is to store samples with a degree of biological activity. Does your loading dye have detergents, chaotropic agents (guanidine or urea) or  b-mercaptoethanol? If not, a solution would be to mix your samples with Laemmli-like buffers after quantification. These samples will not longer  have biological activity and degradation won't be an issue. 

Hope it helps

Dear Sebastian thank you for the answer. I think there is a confusion about my question. After bradford for sure I add Laemmli Buffer into my samples and then perform western blot. In the first one I see equal loading control but afterwards I store my samples which contain Laemmli buffer at -20 and then repeat the same western with the same samples and the loading control (b actin) is not equal as it was before. It sounds unlikely but an explanation is maybe the remaining biological activity even after the addition of detergent? And how trustworthy is b Actin while every paper uses it as control?

What is the method you're using to reveal your WB? Is it film or detection on an automated device (LiCOR, STORM, GelDOC)? If it is film, you are probably experiencing saturation of the bands so even if you have more ptn in one sample than in the other, it won't show, because both bands will be as dark as it can be. The same issue can also happen on a detector, but the dynamic range of those is usually higher (specially if you're using fluorescent antibodies, instead of ECL), so it's less likely.

Maybe when you run the same sample a second time your are running less or some of it degraded due being too much time at RT, and then you can see the difference.

Are you using Protease inhibitors on your lysis buffer?

In our lab we use ECL and a LiCOR detector, and we saw that for beta-actin anything greater than 15ug of total ptn (on lysate) is already saturated, so 15ug, 20ug, 30ug all look the same at the WB. Most beta-actin controls on the literature are saturated bands that really do not inform a lot about the loading really, but it's used anyway, because, tradition.

It is possible that protein could be degrated, I usually store the samples (the homogenated) without loading buffer and add three types of inhibitor of protease and phosphatse (protease inhibitor cocktail from ROCHE and phosphatase inhibitor 2 y 3 from SIGMA).Other thing could you do is check the protein concentration before the WB.

The b-actina is not the unique endogenous control, I know that other partners in the lab use as control b-tubulina.

I hope to help you.

Best whishes 

Derya,

I don't think your problem in this case is the type of protein you've chosen since the initial experiment gave you equal loadings.  It just intriguing that you don't have the same problem for the other proteins you've tested, which would rule out the other possibility that is precipitation of your protein. This last should affect all the protein pool. I would pay attention to Luiza's comments since use of high titers of secondary antibody or film overexposures can trick you showing equal bands by unspecific adsorption and signal saturation respectively.

I routinely collect samples of cells growing in substrates that later are in higher amounts than the proteins of the lysate itself masking Bradford quantifications. In these cases were quantifying is useless I use to run a little amount of my samples and blot them against actin and b-tubulin, then I quantify optic density and adjust the samples accordingly. Isn't the fastest but it helps in some cases.

Depending the experiments I normally use GADPH, f-actin, bIII tubulin (tuj) or alpha tubulin.

 I always store the samples (the homogenated) at -80°C with protease inhibitor cocktail from ROCHE and phosphatase inhibito. Furthermore, if I have to use the semples after 1 week I prefer do again Breadford Assay.

About the control you can use different, depending on whether you have mitochondrial (ex. cytochrome C oxidase), nuclear( ex. lamin B1, Histone H1) or cytosolic (ex. GAPDH, b-tubulina, b-actina...)  protein extract..

Note that Actin proteins tend to be present in cells at very high concentrations, and thus it is important to adjust the loading volumn and Western blot protocol to detect any change in actin protein levels. 

Best whishes

For my blots I load 20 microgram protein per sample and do the detection with ECL system. Before mixing samples with Laemmi buffer, I keep my samples with SERVA protease and phosphatase inhibitors, but I don't add inhibitors in laemmi buffer if this is what's not clear. I really appreciate the comments which were very productive. Next time just out of crusty, I will try b-actin control with less amount and try to store samples in -80. And of course I will let you know the results. Thanks :)