I've been using beta actin and also developed some tests with GAPDH in order to normalise my target proteins. However, since my animal model undergoes to hypoxia, as well as changes in muscle fibre metabolism, these both controls may not be suitable for my experiments.
Some authors use B actin or GAPDH in their studies for similar animal models; but when I run the statistics, it is clear that the signals I obtained from the protein bands are varying significantly among the groups. In other words, my control appears to be unstable and is affecting my final results.
In this way, does anyone have suggestions about loading controls for skeletal muscle samples and could share your own experience regarding these controls?
Thank you very much :)
Hi, it is a very importat issues. You can find a lot of discussions here on reserchgate on this topic.
I suppose you need a loading control for whole extract because you didn't specify if you have to analyse a nuclear, membrane or cytoskeleton protein, and you have to pay attention to the loading control can be affected by hipoxya. So keeping out actin and GAPDH, that you just analyzed, the other used in total extract and in muscle were, normally, alpha and beta tubulin, cyclophilin B, Cofilin or Vinculin. From a briefly research on web probably the cyclophilin B is affected only in smooth muscle, ad I can't anything on alpha tubulin but with beta, but you can have problems with the other two. You have to make an accurate literature research about these possibilities but as pointed out in this really interesting article: https://www.researchgate.net/publication/274728173_An_analysis_of_critical_factors_for_quantitative_immunoblotting
It is better if you choose more than one loading control.
Naturally there are all the news strategies of total protein visualization, compatible with western, from the new Stain-Free Technology of Bioad, to SYPRO Ruby (even if it is better if you have at least a Chemidoc, maybe someone that used this strategy can answer you better!) or the old Ponceau S. The last on it is not expensive, even if less sensitive, and you can decided to compare antibody and total proteins staining.
Article An Analysis of Critical Factors for Quantitative Immunoblotting
Hi Aline,
I haven't used primary tissue in westerns and even a lab that I know that does extensive animal research struggled with loading control. They often used RT qPCR and microchips to analyse gene expresssion. I think one of the cheapest and reliable way is to do a general protein expression as pointed by Asif (Coomasie) or if you want to be sophisticated try silver staining. Please let us know how you overcame this issue by posting it here! Best wishes!
-ED
P.S. Like Valeria said tubulin could be great too. It is one of my favorite loading controls and used it for almost all my blots in this paper (https://www.researchgate.net/publication/260112146_MTORp70S6K_signaling_distinguishes_routine_maintenance-level_autophagy_from_autophagic_cell_death_during_influenza_A_infection OR http://www.sciencedirect.com/science/article/pii/S0042682214000117). Both alpha and beta worked well. Good luck!
-ED
Article MTOR/p70S6K signaling distinguishes routine, maintenance-lev...
Dear all, I am really grateful for your contributions.
I do appreciate the papers you had suggested (I am reviewing them now) and I believe to discuss this topic here was productive and stimulating, indeed. I should start a new phase of experiments this week and once get new results, I can come back to discuss the adopted protocol with you. Again, thank you very much!
Dear Aline,
I have used b-actin (Santa Cruz) for most of my experiments.
Try to use 15 or less micrograms of protein in each well and 1:10.000 dilution of secondary Ab.
Your load can be not good due some problem of incubation. I solved all of my problems with the b-actin when I decreased the signal produced by protein excess and secondary antibody.
good luck!
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