Greetings, we ran gel electrophoresis in the lab on pig red cells ghosts, the first column is the maker.
The second and fourth columns are of concern. The second column contains ghost cells, the fourth column contains inside out vesicles. The band indicated by the right arrow travelled faster than the band indicated by the left arrow, they should be the same protein (Anion Exchanger 1 from pig blood).
Any suggestions would be very much appreciated.
Thank you!
Hi Rafael,
I think a little more information is required for a full answer but I will help as best as I can. Which gel and stain are you using?
From looking at the band it looks like you have either a much higher amount present or an oversaturated signal. I would either dilute the sample or place less in the well. From the looks of it, the size of the signal is what is making it look as if it has travelled further. if we compare wells 2 and 4 to other wells on your gel we can see that the bands line up with others on the gel, especially those expressed in the middle of the gel in wells 3, 4, 5 and 6. Following the middle bands we can see that this is also expressed in well 2 but at a much lower level (this looks to be the same for the band with the arrow on well 2).
I would suggest diluting the sample of well 4 and re-running the gel, it seems there is much more present in that particular well. Also some stains are very sensitive and therefore when analysing samples different sources may require more dilution to prevent oversaturation.
I hope this helps.
Hey Rafael,
SDS-Gels often run uneven and the difference in your gel is rather small. So i would not be too concerned :) SDS-PAGE is also more an estimation and not a very accurate method to determine the size of a protein.
It also looks like that the concentration in lane 4 is much higher compared to lane 2. High concentrations -> strong bands often lead to the impression of "faster" migration.
You could try to dilute the sample and run another SDS-PAGE or you can use other techniques to make sure that these are the same proteins (e.g. mass spectrometry, western-blot etc.)
hope this helps.
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