western blot for exosomes sample. I used antiGAPDH and antipalladin antibodies.
I load 20ug of sample, in 1xRIPA buffer and 4xLaemmli buffer. Images 4&6 same membrane.
hi,
May I know which type of membrane? what is the detection have you applied? maybe also you touched the membrane by your hand make it not clean
Hi Ekhlas,
Thank you. No, I did not touched it by hands, I used forceps. It is a nitrocellulose membrane.
I used antipalladin and anti gapdh antibodies.
as general, see this website, maybe it will help to recognize the problem:
http://www.abcam.com/protocols/western-blot-troubleshooting-tips
Hi Zainab, I don' know if you resolve your problems yet.
In any case I want to ask you if the first image is GAPDH. I don't have experience with exosomes but I known that you waited a good signal, at least, for GAPDH. In all the three I saw a patchy background, so I ask you:
- Did you control the transfer with Ponceau? First you have to exclude any problem in this step
- Which block did you use? In which solution did you dilute the antibody? Did you try to vary the diluition concentration?
- How may times did you wash the membrane? Did you use tween
- Did you incubate the antibodies ON or for few hours? Did you control that your membrane was completely covered? A lot of times the patchy background it can be caused my not covered membrame, the same when you used ECL
- The last two photos (same antibody?) are negative because you obtained this ghost aspect after film exposure? The ghost aspect can be caused by residual background noise or excessive signal
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