I need to use a loading control for Western Blot that neither includes a cytoskeleton protein or a protein that interacts directly with the cytoskeleton. My issue is that I incubate cells with methylmercury which has been shown to induce alterations in the cytoskeleton. I am afraid that by using one of these cytoskeleton proteins as a loading control I may be detecting alterations in my target proteins that do not exist since cytoskeleton proteins may be altered or vice versa.
Hi,
The first thing to believe in is to stain the membrane with ponceau red or amidoblack to know what you have on your membrane after transfer.
I've never been a fan of e.g. checking loading at 37 kDa with GAPDH to normalize abundance of a big glycosylated membrane protein of 120 kDa that is 4 cm away on the membrane. (and of WB quantification at all which is another question)
As Krishna kumar Ganta says, the use of housekeeping proteins in a context of cellular stress can be misleading. For example I remember people of the circadian rhythm field stop using actin because it turned out to also rhythmically fluctuate.
You can take a picture of the stained membrane or scan it and do whole lane densitometry or around the region you blot with the antibody of interest.
I've heard such things were asked more and more often by journals and reviewers, don't know if it is true.
An example of the reagents Michael Okpara mentions would be stain free from biorad (I had seen ads for it but never tried it though).
Good luck
A little more detail would be incredibly useful: you don't even specify what cell type you are using, let alone what species.
Beta actin is a common loading control (as are tubulins), but GAPDH is a fairly traditional option, if cytoskeletal components are to be avoided.
You could always just search the literature, or better yet: exploit companies that have done that for you.
https://www.bio-rad-antibodies.com/western-blot-loading-controls-antibodies.html
Cyclophilin A is an option, or vinculin (we use vinculin for muscle western blot normalisation).
Your concern regarding the use of cytoskeleton protein as a loading control in this case is correct. I would recommend that you use GAPDH as it is stably and constitutively expressed in all cells.
Another option is to use histone 3 or you could quantify total protein amount and use that as a loading control
Your experiment involves cellular stress, meaning that each an every compartment in the cell exhibits certain level of stress. First determine the stress in different cellular compartments. And chose a protein from unaffected compartment. Stress includes cytoplasm, ER, mitochondria nucleus etc. If you use cancer cells for your experiment house keeping genes tend to increase in expression. I suggest having dual controls. Gapdh as suggested by others and pick one more protein like ribosomal proteins which are stable. Or beta 2 micro globulin can be also be stable. You can consider ratios when in doubt.. Good luck
Hi,
The first thing to believe in is to stain the membrane with ponceau red or amidoblack to know what you have on your membrane after transfer.
I've never been a fan of e.g. checking loading at 37 kDa with GAPDH to normalize abundance of a big glycosylated membrane protein of 120 kDa that is 4 cm away on the membrane. (and of WB quantification at all which is another question)
As Krishna kumar Ganta says, the use of housekeeping proteins in a context of cellular stress can be misleading. For example I remember people of the circadian rhythm field stop using actin because it turned out to also rhythmically fluctuate.
You can take a picture of the stained membrane or scan it and do whole lane densitometry or around the region you blot with the antibody of interest.
I've heard such things were asked more and more often by journals and reviewers, don't know if it is true.
An example of the reagents Michael Okpara mentions would be stain free from biorad (I had seen ads for it but never tried it though).
Good luck
You can try GAPDH, which is used as loading control for whole cell and cytoplasmic extract. But, you have to ensure that your experiment does not have any effect on GAPDH or else you can try dual loading control.
I recommend GAPDH. Because in some circumstances, beta-actin is utilized in cellular metabolism for specific functions especially under stressful conditions. This could show a slight variation in quantification with control. GAPDH does not have multiple interactions to show variations in the cell.
I would recommend also in this case GAPDH as loading control not beta-actin .
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