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
Hi Valeria,
Many thanks, the problem is resolved by rewash the membranes and rescan them.
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