This is our first time doing SDS-PAGE, and therefore we would like to hear from more experienced people if we interpret the results right.
Attached is a picture of one of our gels with our positive and negative control. These are from E.coli cells where on produce amyloids/curli (positive, P) and the other one is modified not to produce amyliods/curli (negative, N). Both controls have been through an isolation process to try and isolate only amyloids. Then some has been treated with fomic acid (P+ and N+) and some just with MilliQ water (P- and N-).
Here is our interpretation:
The formic acid-treated positive control, P +, shows a clear band between 14.4 and 18.4 kDa. This band is not seen in the sample with MilliQ water, P-, or the negative control. Thus, the band is expected to be amyloid monomers, as the molecular weight also corresponds to CsgA in curli, which is thought to have a molecular weight of 13-17 kDa. The thickness of the band may indicate a high protein concentration. In addition to this clear band in P +, several less clear bands are seen through the gel that can give an effect of smear. This suggests several different sizes of proteins have been present in the sample (not successful isolation). Also several clearer bands is seen for P + compared to P- (this we don't understand why).
In the formic acid-treated negative control, N +, several less clear bands are seen throughout the gel. This is due to proteins of many different sizes in the sample, as seen in the positive control. Also bands in N+ appear more clear compared to bands in N- (why?). In general, several identical bands are seen for both controls. This is to be expected as both controls are E.coli and therefore the same proteins are found in both controls except amyloid proteins in the negative control.
Generally there is a tendency of diffused bands, which can give a smear-like effect. This may be because the temperature during electrophoresis has been too high. A solution may be to run the electrophoresis for a longer time at lower voltage.
Please let us know if this is correct, if we missed something or other thoughts :-)
Thank you!
The explanation given by you seems rational. However, to seek more information, perform MALDi TOF of thick prominent band.
- Badges
- Science method