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
Hi Hiba
many possible reason for that:
1- the amount of protein was not enough to give you the signal that you need.here can I ask you how many cells you were seeding lets to say 1 million cells or 2 million cells would be more than enough( here you can load 30ul in each well of 10 well comb or 15 ul into 15 well comb).
2- or may be the transfer was not working well in which the protein bands still in the gel and not transferred to the nitrocellulose membrane( In this case you can stain the membrane with ponaceu red if you got band this will tell you that the the transfer is ok).
3- the secondary antibody has to be from the same as the primary for example if you are using rabbit the secondary rabbit( some time mixing can happen here).
If you have any further questions feel free to ask
Shilan
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