I have been doing western blotting for past 8 months. i am not able to get consistence result of my needed protein .by sometimes i get distorted band and sometimes ok and sometimes i don’t get any band at all. but every time i do i have b- actin. i had one question. if my lysate preparation step is wrong do I see b actin band or not? or if sometimes if unknowingly I use cell debris in cell lysate do I get b actin bands or not?And whether it is due to the degradation of my needed protein in environment? I use protease inhibitor and PMSF and I do all cell lysate preparation in ice. Do you think that something is wrong with my cell lysate preparation?
I will first check your primary antibody against your proteins. Since you see the band sometimes, it may not be issue with your lysates. Do you reuse the primary antibodies for Western? To test whether it is the problem with your lysates or antibody, I will use another antibodies other than house keeping gene actin. I would also stain my membrane with Ponceau S stain to see how much protein is transferred.
Hope that you can work this out.
Hello
do you quantify your protein first ?
Look at the discussion in GR & Links
Hope help you
Good Luck
https://www.researchgate.net/post/No_consistency_result_in_western_blotting2/1
http://info.gbiosciences.com/blog/bid/130848/7-Common-Problems-with-Western-Blotting-Solved
Thanks for the reply. Chan thu I reuse the primary antibodies for 5 times. I also check with Ponceau S stain whether it has transferred or not. But my lab mate also uses same antibodies her results are good. I have attached by results below can u evaluate it.
Thanks for the reply.Professor Saleh alkarim I do the quantification of protein every time. I load the same amount of sample. I have attached by results below can u evaluate it.
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