Hi everyone,
I'm having problems with my western blots. I'm analyzing spinal cord tissue after injury and treated with compounds that regulate cell cycle and protein synthesis. Proteins were isolated using RIPA and measured using BCA assay.
I also used stain-free gels from Biorad, to confirm loading (picture 2 and 3), but my boss does not wish to use the gels for protein normalization, it has to be some housekeeping protein. Beta actin is unequal between samples, even in the same group (picture 1). In literature there are examples where beta actin was used without problem in the same settings. I looked into other loading controls, but I would like some recommendations first.
Do you think the problem is in the expression of beta-actin, or somewhere else? Should I try different loading controls, or change something else?
Thanks for any advice.
I think that it is great that you are using the stain free gels and the new BioRad system. We recently purchased one and really think that it works well. We had a reviewer specifically ask about total protein staining. This, in my opinion, is the most accurate and reliable method of standardizing protein. It does not change due to biological conditions. It is literally what was transferred from the gel to membrane. In theory loading controls such as beta actin or beta tubulin do not change or should not change. However, this might not always be the case and therefore would not be a reliable method of standardizing protein. I looked at your blots and the level of beta actin does not directly correspond to the total protein transferred. For instance, you first 3 lanes appear to have had almost equal total protein transferred. However, your second band for treatment A is way lower than the other 2 bands. I would run either beta tubulin or GAPDH on your samples to see if you get similar results to beta actin.
There are a lot of posible explenations. It could be pippeting error, gel quality and even mistakes when you quantify. I suggest you to be extremelly carefull in this aspects and if this does not work try whit gapdh or another housekeeping proteins.
I think that it is great that you are using the stain free gels and the new BioRad system. We recently purchased one and really think that it works well. We had a reviewer specifically ask about total protein staining. This, in my opinion, is the most accurate and reliable method of standardizing protein. It does not change due to biological conditions. It is literally what was transferred from the gel to membrane. In theory loading controls such as beta actin or beta tubulin do not change or should not change. However, this might not always be the case and therefore would not be a reliable method of standardizing protein. I looked at your blots and the level of beta actin does not directly correspond to the total protein transferred. For instance, you first 3 lanes appear to have had almost equal total protein transferred. However, your second band for treatment A is way lower than the other 2 bands. I would run either beta tubulin or GAPDH on your samples to see if you get similar results to beta actin.
I suggest you have a look into the following article:
Rocha-Martins M, Njaine B, Silveira MS. Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development. PLoS One. 2012;7(8):e43028.
Thank you all for valuable suggestions, I will update the tread once I acquire and try different loading controls.
I would like to update the loading control problem- I tried beta-tubulin loading control, since I read that it should be more appropriate for spinal cord injury tissue. I got a result that makes even less sense, so I do not believe housekeeping proteins are reliable loading control in some cases. I attached the result from one membrane where we detected firstly beta-tubulin and then beta-actin. It clearly shows that these two are not expressed at constant rates in our samples and it is not a case of bad pipetting or protein degradation.
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