A big challenge when running western blots with primary material (e.g cancer material or leukocytes from individual blood samples) is the biological variance in the expression of traditional loading control markers such as action, tubulin etc. They simply vary to much between individuals to provide meaningfull information about the loading of the sample. In such cases, coomassie staining of the gel or ponceau staining of membrane should, at least in my mind, be regarded as an adequate substitute for the traditional loading control.

Hi,

I have frequently used Pancheau S stainings after transfer to the blot to a) see if the transfer has worked and b) to visualize the lanes so I could cut them into strips to test with different antibodies at the same time.

It works great, just put it in for a few minutes and when you're done, wash it with a few rounds of either PBS or wash buffer. Doesnt affect readout using fluorescence or ECL as far as I could tell.

Erik

The best loading control in western blots is a housekeeping protein (actin, GAPDH, TPB, etc) after striping the same membrane. If selection of a good loading control turns difficult, I would use ponceau red staining (because is the same membrane) rather than coomassie blue.

OHH is correct. Use an internal control or reference protein. Publishers will ask for it. This way you are normalizing twice. Total protein loading is consistent and relative to an internal reference.

you can use ponceau stain after transfer to know your sample run equaliy, however you will need to use reference loading control protein such as..Actin, tubulin normally used by most researcher.

there are pre stained markers which transfer to blot. They are more reliable than staining.

I agree with Subhendu that Ponceau staining of the blot is great for monitoring transfer, but you do need a control for the probing procedure, especially because Ponceau staining is not useful for quantitation. However, I have to take issue with Randhir's suggestion that pre-stained markers are more reliable for monitoring transfer than is staining. Pre-stained markers can appear completely transferred when less than 50% of total protein samples are transferred. If you stain your gel with Coomassie after blotting, you may be surprised how much is left in the gel. This is especially true as the amount loaded increases, and is one of the reasons why loading controls are so important.

Ponceau is a very good way to quickly check loading control before blotting, so you can see if the gel needs to be run again. I think usually this is enough without the housekeeping proteins as you do see Ponceaus appearing as loading controls in some major publications. e.g. http://www.nature.com/cdd/journal/v18/n3/fig_tab/cdd2010126f1.html#figure-title

Using Ponceau staining you can make a copy of your stained blot, e.i. on a overhead slide. So you can place the protein stain later on your immunostain. Disadvantage: not very sensitive. Advantage: completely removable staining.

Coomassie has the disadvantage that is is not removable from the membrane without destruction of the antigens.

Indian Ink is a quite sensitive, but also not removable alternative. Sensitive enough.

The best way to get some more insights: arrangement of samples: e.i. protein ladder, control, sample ...

The NC-membrane has to cut into strips which will be stained later on by different methods: Protein stain, specific stains for glycosylated proteins are available (e.i. biotin-labeled lectins, oxidation methods, ...), immunostains (even in combination: on antigen detection with an HRP-labeled antibody, detection of the second antigen simultaneously with an AP-labeled, only the color reaction has to run separately).

A big challenge when running western blots with primary material (e.g cancer material or leukocytes from individual blood samples) is the biological variance in the expression of traditional loading control markers such as action, tubulin etc. They simply vary to much between individuals to provide meaningfull information about the loading of the sample. In such cases, coomassie staining of the gel or ponceau staining of membrane should, at least in my mind, be regarded as an adequate substitute for the traditional loading control.

Whilst Ponceau is useful (I use it every time) it only really helps you to know if the membrane you are using has a good protein transfer or not or if you have bubbles in your transfer sandwich. It is not sensitive enough to be used to quantitate, and so most people use actin, tubulin etc.

André makes an excellent point. One should be careful when relying on an antibody that detects a "loading control protein" for relative quantitation. Depending on the experimental conditions, the expression level of a particular housekeeping gene may change. There are a number of publications in the literature that demonstrate this experimentally and I advise you to look them up.

You can certainly attempt to stain the protein gel post-transfer with Coomassie and also stain the membrane with Ponceau to check if your loading control is staying at a steady level; I usually use amido black, which is more sensitive and does not wash away as easily as Ponceau, although it will cause the membrane to fluoresce if you are using Li-Cor detection and is hence suitable only for ECL/film. Spyro Ruby Red might be a better stain than Ponceau if you need to avoid a blue stain like amido black. There are also several studies in the literature about using total protein stain for quantitation purposes.

In summary, I propose that both a loading control and total protein staining should be used in combination for relative quantitation of a protein by immunoblotting.

Simply google "ponceau S"and "loading controls", you will be able to find published papers suggesting ponceau S as an alternative and better method. If you prefer housekeeping genes, actin should be avoided, to my knowledge.

Here is one

Anal Biochem. 2010 Jun 15;401(2):318-20. doi: 10.1016/j.ab.2010.02.036. Epub 2010 Mar 3.

Reversible Ponceau staining as a loading control alternative to actin in Western blots.

Thanks Bojuan... :-)

Agreed, both actin and GAPDH can vary in their expression levels depending on culture conditions. There are number of published examples of both in the literature. They can still be valid loading controls under suitable experimental conditions, but a second control should be mandatory before relying on either of these alone.

Tubulin mRNA levels are also known to vary during the course of the cell cycle in S. cerevisiae although the levels of alpha and beta tubulin protein appear to be stable.

Two more links of interest for those following this question:

http://download.springer.com/static/pdf/972/art%253A10.1007%252Fs12033-013-9672-6.pdf?auth66=1383141155_b5c62d1e048eac1084700a16db89d021&ext=.pdf

Bio-Rad apparently sells a stain-free gel system for protein SDS-PAGE. I don't know how well it works though!

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6360.pdf

Hi everybody,

I agree with the fact than Ponceau staining is suitable as loading control. But I have one question.

Do you have to use all the lane? Can you use only one band, for example, the 40 or 25 kDa kDa one? I found a paper that do it

Journal of Neuroendocrinology, 2005, Vol. 17, 255–261

Thanks!

I am in complete agreement with most of the comments on this thread.  I would recommend avoiding coomassie staining as a means of measuring loading (unless performed in parallel), as any stain in methanol/ethanol/isopropanol will obviously result in fixation of samples and difficulties in downstream processing.

It is my experience that "loading controls", or selected "housekeeping" proteins are unreliable.  There is too much variability in expression among different cell types, patient samples, etc. for them to be used as a reference standard in western blots.  I have also found that many of the antibodies available, do not reliably report the concentration of the loading control due to saturation issues (you should test this for yourself).  One should stress that typically, "loading control" proteins are much more abundant than the protein of interest, and saturate membranes at concentrations required for the detection of low abundance proteins.  Therefore, it is better to use "total protein normalization" as means to assess loading.  Ponceau S, Sypro Ruby, and Bio-Rad's "Stain-free" technology are best for this purpose, as they show linear signals over a wider concentration range than "loading controls".  Additionally, all antibodies used for western quantification should also demonstrate linear signal response over the total protein loading concentration range employed in your studies.  This latter issue is not trivial, and should be a major concern for all researchers performing quantitative westerns.

I recommend the following video to my student's and colleagues.  Credit and thanks should be given to it's author, Aldrin Gomes. It's an eye opener!

http://www.youtube.com/watch?v=zrfAHAo4xT0

check this paper ..

Thanks Sanket...

To clarify in the light of Jon's comment, when I said "You can certainly attempt to stain the protein gel post-transfer with Coomassie", I should have indicated that doing so is most useful when there is a second gel that is not subject to the transfer process for comparison/control. To echo Lucy's point, you'll be surprised at how much protein remains in the gel, but more importantly, you'll get an indication of aberrant transfers across the width of the gel sometimes!

Point of interest: I've also learnt from my former advisor that the methanol fixation during amido black staining was the only way he could preserve a protein of interest to him that would otherwise not show up on his blots. Amido black, however, is is a reversible stain unlike CBB, which should not go on a blot unless you are done with it.

Jon, those are excellent points you've made concerning loading controls.

Thank you Mark for the information....

Now a days I am using the Stain Free gel tech. by Bio-rad. And its working fine.

Great to hear Pankaj. I've come across that technology and I'm glad to hear it's working well for you.

Dear Pankaj,

I also faced the same problem for  housekeeping gene in cell membrane fraction during colonocyte differentiation. I stained the PVDF membrane by Coomassie after developing the desired protein. You can try that.There is a beautiful paper on this, which I followed.

http://www.ncbi.nlm.nih.gov/pubmed/21186791

I got many new insights from several researcher through Researchgate, you can follow that thread regarding housekeeping genes. https://www.researchgate.net/post/What_are_proper_housekeeping_genes_in_the_plasma_membrane_fraction_for_western_blotting

All the best,

In my case, I work with several mutant and their protein expression profile are totally different. So Ponceau S is suitable for checking proteins on membrane after transferring but not for loading control. For loading control, I use house keeping genes. 

For what it's worth, I've had no problem using Ponceau S as a post immuno method to quantify total lane protein on NC or PVDF since I have no downstream requirements for the proteins after that. I am now using colloidal coomassie post-immuno on PVDF as it happens to fluoresce under 685nm stimulation making it very easy to use on NIR scanners such as LICOR and others. I agree Ponceau may not be super sensitive but feel it is more fool proof than using a single highly expressed "housekeeping" protein as a loading/transfer normalizing signal. It's also easier not needing to run a second antibody. Many reports doing controlled tests of various methods are easily found on pubmed, as others here have been linking in.

With regards to so called "stain-free" gels at Bio-Rad, I have used these and found them helpful for initial validation of gels. However the UV exposure had some inhibiting effect on complete transfer for me and I gave them up after I had optimized transfer conditions.

Another detail about NIR imaging that might be useful. Rather than staining gel to check on blot quality, using a NIR 700nm image of the blot, simply enhance the brightness and one can see any bubbles or dehydration related "bald" spots before continuing with immunostaining.

The JBC also recommends normalizing using total protein loading:

http://www.jbc.org/site/ebm/orientation/2017/AE%20Slides/Slides_%20Efforts%20to%20improve%20transparency%20by%20Roger%20Colbran%20Associate%20Editor.pdf

There's a new paper in press at Molecular and Cellular Endocrinology that evaluates total protein staining as a normalization method for Western blot analysis.

Article Use of the REVERT ® total protein stain as a loading control...