On imaging, there was very faint lines. I am not sure they considered bands. I used beta actin and p-53. Is it possible that cell lysis not worked at all? I did BCA and got reading for the proteins so how can BCA give reading and western show no result even for beta actin?.
Hi Heba,
It could be a cell lysis step but also it could be a western blot issue. If you give us more info about how did your gel running and transfer go, this will be really useful.
For instance, did you stain your membrane with Ponceau red stain after the transfer? did you use a pre-stained protein marker? and so on.
good luck
Hi Heba,
Many factors can interact and make your western.B fail. So you need to provide more information.
1-Do you have smear? (like many non specific faint bands a part from your protein size band?). If yes, then your protein could be degraded, you need to make sure to add a protease inhibitor (for example PMSF)to your lysis buffer (for example RIPA buffer). Also you need to confirm that the protease inhibitor you are using is fresh (avoid showing and freezing, try to make aliquots or pick up the exact required volume and return it to -20C right away. Always use just the volume enough for your experiment.
2-Make sure the pH of your running buffer is ok. If it is commercial, it is the best. If you prepare it on your own, you might need to take the pH seriously (depends on your protein am not sure about p53 in western blot to be honest)
3-Are your bands whitish? or sometimes white? if so, you need to decrease the exposure time when you reveal by chemiluminescence reagents..
Best of luck!
you did not mention how you prepare your protein cell lysis sample
4-make sure you mix it with ration of sample buffer/beta-mercaptoethanol (9:1) then heat inactivate it by 5minutes incubation @ 90C before running your gel
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