loading control issue in blot with H9c2 cell protein, pleeeeeasee help - (Feb/25/2014 )
I am working with rat cardiomyocytes (h9c2) and I am new to these cells. After different treatments, when i do a western blot, surprising or rather I may say, shockingly i am getting loading controls such as GAPDH and beta actin always irregular/upregulated!! I thought at first its my issue with loading, but even after caring to minute details Still Iam having problems with these controls. Papers say GAPDH /actin is fantastic!!
yesterday I performed the western again, GAPDH and beta actin as usual upregulated in treated cells, but I found some non specific band near 60-70KDA in uniform fashion!! very perfect!!
Now I doubt if my loading is not proper how come these band come uniform? What might be those bands? I thought hsc70 at first, but cardiostress may normally affect the expression pattern of hsc, right? If I Know what is this protein can I use it as my internal control?
Is there anyone working working with h9c2 cells in our forum who can help me? please help...Iam stuck with it for past few months and need to settle this endogenous loading control issue once if I want to progress into my main project...
Thanks in advance for any suggestion
It is not uncommon to see housekeeping protein expression level varies in response to different experimental conditions such as treatments, development stages, gene manipulation, etc. You may also have observed that housekeeping protein detection in western blotting is often saturated due to a combination of its high-abundance and a sensitive detection method (antibody-based immunodetection).
These problems can be solved by using total protein loading control. I have seen many papers on JBC presenting a coomassie blot or gel image for loading control. But coomassie staining takes a long time and one may need to run two gels (one for loading control coomassie staining, one for blotting). I also heard that people stain the blot with coomassie after immunoblotting to confirm the total protein loading. Either ways, it is tedious and need extra work.
Bio-Rad provides a good solution to the problems. They have a stain-free gel and a so-called V3 western workflow that allow researchers to visualize and measure proteins in gels and on blots without any additional steps added to the western procedure. Total protein loading control can be measured either from the stain-free gel image or the stain-free blot image and used to normalize the target protein data.
Please check out the following publications for more information:
1. http://www.jove.com/video/50948/v3-stain-free-workflow-for-practical-convenient-reliable-total?status=a52954k V3 stain-free workflow for a practical, convenient, and reliable total protein loading control in western blotting. Posch A, Kohn J, Oh K, Hammond M, Liu N. J Vis Exp. 2013 Dec 30;(82):50948. doi: 10.3791/50948.
2. Stain-Free total protein staining is a superior loading control to β-actin for Western blots. Gilda JE, Gomes AV. Anal Biochem. 2013 Sep 15;440(2):186-8. doi: 10.1016/j.ab.2013.05.027. Epub 2013 Jun 6.
3. Comparison of Stain-Free gels with traditional immunoblot loading control methodology. Colella AD, Chegenii N, Tea MN, Gibbins IL, Williams KA, Chataway TK. Anal Biochem. 2012 Nov 15;430(2):108-10. doi: 10.1016/j.ab.2012.08.015. Epub 2012 Aug 26.
Hope this helps.