Hi Mohamed, I never used HaCat cells, but in many different cell models I have used I have seen always a single b-actin band.

What antibody are you using? I use Neomarkers Ab-5 antibody http://www.thermo.com/eThermo/CMA/PDFs/Product/productPDF_56260.PDF and it works pretty well.

The two bands you are getting might not be of same intensity and probably your beta-actin antibody is polyclonal so the 2nd band is coming because of non-specific binding of the antibody. Try to mature your antibody if its polyclonal by repeatedly using it for 3-5 blots (don't throw the antibody solution after the blot). Preserve that solution in Sodium Azide standard concentration at 4'C while repeat analysis. You may get good results after the antibody is matured. Secondly, and most important thing is that you must try a much lower dilution of your antibody like if you are using 1:500 now then try with 1:2000 or 1:5000 dilution. Good luck

From my personal experience, pH of the gel buffers as well as running buffer are important players in such type of event. I was also face the same problem. After maintaining the pH of all solution properly, I got the single band.

You might want to try a monoclonal antibody to b-actin which will give you a clean one band. I use one from Sigma and I always get one band.

I also have no problems with a monoclonal anti-bactin from SIGMA. It could be a thecnical problem, if you develop with ECL and films, since b-actin develop very rapidly, if you move a bit your film it gives you 2 bands (but actually its only one band). You can stripping your membrane and incubate again with the antibody to verify if it is the case.

Hi Mohamed. I used two antibodies for b-actin: one monoclonal from Sigma (cod.A4700) and one polyclonal from Cell Signalling (cod. 4967). In both cases i dilueted them 1:1000 in 3% BSA/ PBS 0.02% sodium azide and always got a single band.

i use monoclonal antibody from Santa Cruz company. i do not use polyclonal antibody. i tried several times but two bands. my lab mates also using same antibody and also getting one band not two bands

I see...your problem could be in ECL..try to avoid minor movements of film while exposing (when keeping the film over blot, while closing or opening the cassette etc). If you're not using ECL and getting twin bands..then it could be post-translational modification of actin..

Are they working on the same cells what you use? I too use Santa Cruz's antibodies. what kind of detection system you apply? I use BCIP-NBT system and it works good. If you are using ECL system then I think Ermelindo Leal's doubt could be right... Good luck

To make it simple, I would ask labs around yours an aliquot of other actin antibody and test the membrane again.

To make it complicated get some recombinant b-actin, or peptide of the antibody, to incubate the antibody with in order to check the specificity of the antibody.

I would like to raise other possibilities. Did you reuse the antibody? Or, did you strip the membrane before blotting with b-actin antibody? b-Actin antibody works so well so some lab will reuse it which may give double bands. If the membrane is stripped after another antibody is will be common to see two bands.

We do not reuse antibody in my lab

Hi Mohamed

If you could post a pic of the gel then it would allow us to better diagnose the problem.

What size is the second band? It is highly unlikely to be a posttranslational modification of b-actin. It is possible that there is some non-specific binding from your monoclonal (after all, you have bought from Santa Crap), and also it is possible that it is carry-over of denatured bovine H+L Ab chains from residual FCS in your lysates (these migrate at approx 30 and 50kDa, however will only show up if your 2* Ab cross-reacts with bovine).

From what you describe (the intermittent appearance of the band and the fact that no one esle see's this) it sounds highly likely that the real cause is technical in nature and occurs during your membrane placement on your gel. The instant you place your membrane on the gel, some protein is transferred. This is especially evident with highly abundant proteins such as b-actin. You can test it if you like (I certainly have!), and with a little creativity you can even make pretty pictures this way ;)

Best of luck.

Al

Hi Mohamed,

There are three potential reasons for this

firstly as jinesh said you may have moved the filter filter during ECL, but it would be bad luck if you moved the filter up or down precisely-when I am clumsy and I do this, the secondary band usually moves up or down and side ways as well so the bands are not usually displaced precisely vertically. Also if you have another blot in the cassette do you get additional bands in that as well?

Secondly it could well be secondary modification- actin is modified (most likely in my opinion).

Thirdly if you re-use your membrane then it could be carry over from the previous blot.

Finally, like Tiff T I quite often see a secondary band on actin blots (I use I19 santa cruz goat antibody), but mostly if I expose the film for a very long time because the blot is in the cassette with other membranes. This is a far more problematic for your interpretation because I would suggest that the main bands themselves could be overexposed, and the signal will be saturated. If you are using the actin blot to normalise for loading (especially if you then do densitometry for quantitation) then you might be assuming you have even loading. If you only see the 2nd band after a long exposure then I would suggest that that is the problem, and you should just do a light exposure. The actual identity of the 2nd band will probably never been known, and is just a result of cross reactivity, and I wouldn't worry about it too much.

I have been using sigma b-actin ab for long time and used to get only one clear band, but more recently, with the same ab (with treated MCF7) I got two bands while with the control I still get one clear band, I thought maybe the treatment caused the b-actin to degrade. Today it was our departmental seminar, the presenter blotted the membrane with b-actin and interestingly only the healthy cell(positive control) have two bands while all other cancer cells have one bands only. So, is two bands of b-actin related somehow to healthy cells vs. unhealthy cell?

Its because of shaking of your xray film while exposing, beta actin gives very high reactivity so you have to be very quick in exposing without shaking..

I see something similar with b-tubulin in smooth muscle cells (but not in other cell lines). So it could be cell type specific? I use licor scanner not ECL so duplication is not an issue, and I don't reuse antibodies. It may be post-tranlational modification (I use protease and phosphatase inhibitors in lysis buffer). One reason you see it some time and not other could be the distance you run the gel i.e. do you always run it far enough to separate out two bands. Also how much you "over-develop" the blot may impact, the lighter the bands, the more likely you are to see two? I am not sure if the actin antibody acts exactly the same as this, just some suggestions. As others have said it could be non-specific interactions with other actin isoforms

By the way, actin is also a substrate of several enzyme

Cleavage of actin by interleukin 1

www.pnas.org/content/93/5/2234.full.pdf

So I would not be surprise to see several actin band if you are detected actin

I have dual bands for beta actin in human serum as well. Differing by under 10kDa. Repeated the visualization twice just to be sure it isn't an artifact due to xray movement. I do note a number of my proteins of interest in serum coming with 2 bands as well. If it matters I had used Cell signalling Tech antibodies for my protein of interest.

We had the same problem several times although we used different actin antibodies, it seems most likely that these are posttranslational modifications of beta actin in some cells. what we are going to do is to use high concentration gels like 15% to have the two bands closer as one band

I read all the answers given here. I'd like to add something. No matter I use fresh aliquot / reuse aliquot of Actin or frsh membrane/ stripped membrane; I sometime observe single actin band, sometimes doublet band in a same cell lysate using same lysis buffer throughout my PhD tenure. :-/ ......I don't know any EXACT reason

(see the attached images in Q7 cell (STHdhQ7/HdhQ7))