H9c2 cells..please 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
actin is a workhorse of cardiomyocytes, so I am not surprised it's changing with treatment. Google what others use as a control. My quick search rendered two papers where they used tubulin and Coumassie Blue staining to cotrol for loading.