Western blotting :In first i could get the lrp1 both bands but now its very difficult to get the bands. instead i get non specific bands .for cross check i have done the western blotting of old sample also i got the result. i know that i have some problem in my cell lysate preparation. What could be the mistake?
If your cells were unhealthy or if your cell lysate preparation was not prepared correctly, this could account for the discrepancy. Did you use protease inhibitors with both samples? Did either sample sit out at room temperature for a long time? I have had a similar problem before if I had a low % viable cells in my culture before protein extraction.
You need a positive control (maybe you have one, but it is not obvious from your description). Do you have one and if so, what is it?
Thanks Jared Taylor for your suggestion. I will see that.
Alejandro Martin : i dont have the positive contol. Will it help me to know weather my process of cell lyste preparation is right or wrong?
Sario Bano : I do the same thing that you wrote. i think i have problem in the cell lyste preparation. And thanks for your reply.
How did you prepare the lysate and why do you think it might not be properly prepared? Are you able to get bands for other proteins with a different antibody?
i had old sample(lysate) left which i did western blotting. The result came and also i had done striping process of the old membrane the result came. But now no result if i do the western blotting from the initial stage. And i haven't tried in other protien till yet only LRP1.
First i wash my sample with chilling pbs for two times(1000ul). and use ripa and pmsf and protease inhibitor. and scrible with scrapper then collect the sample. then put in ice for around 15 mins with inverting for 3-4 times in every 3 mins interval. then centrifuge in 4 c for 14000 rpm for 10mins. after that take out the debris. and do protien assay test. then put sds loading buffer + beta- mercaptoethanol (100ul:1ul) then heat for 5 mins in 95c and put in ice instantly for for 3 mins then and centrifuge for 3mins and then loading.
To be honest your lysate prep procedure seems OK to me. Have you checked the identity of the cell line? Cross-contaminations are surprisingly common.
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