Hello everyone,
Hello,
The picture down blow is the western blot with wired band-shadow, I would like call it like this. They are the bands I want, but I have no idea why they will have two bands close to each other, which I did not see before. Also, the size of band is not equal. Would you guys have some experience about this?
Some notes I can came up for this experiment are:
1. This is 10% gel. And I made new tris buffer (PH around 8.8) for running gel.
2. The electrobuffer I used was from the day before (1* dilution form 10* stock).
3. The blotting buffer I used has been used for three times (Normally, our lab recycled blotting buffer at least for three times).
4. I used constant voltage (90 V for stacking part and 160 V for running part).
5. I also have been reused first antibody for three times and this is my fourth time(still good signal).
I hope those notes can give you guys some clues to see the problem.
Thanks for the time. I am looking forward to comments from all of you.
Another possibility is that the target protein has suffered a modification, such as proteolytic cleavage of a small number of residues from one end.
If you have a look into detail to your bands, not all of them have double band, so, as mentioned before you could have modified proteins or just isomeric proteins. Have you tried to identify the protein from each band?
It may help.
Good luck!
@ Pavel Májek, thank you for your comments. I am less confident about the protein modification, because I had same experiment before and I did not observe such phenomenon. Probably, something went wrong with sample buffer or other technical stuff. I will definitely repeat it again and to see what happens.
Thank you very much.
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