I have observed expression of two splice variants, which should code protein isoforms of 51 and 55 kDa. I have antibody for the protein, but is there any conditions for SDS-PAGE to differentiate between the isoforms with such a small difference in size?
Would 2D-PAGE be better option? The predicted pI are 5.12 for 55 kDa and 4.94 for 51 kDa isoforms.
Hello Mervi,
Unless your gels are of poor quality, there should be no problem in distinguishing two polypeptides with Mw 51 and 55 kDa by SDS-PAGE analysis. I would use a regular Lämmli gel with 12.5 % polyacrylamide, avoiding to overload samples (ideally no more than 1 µg/band, run several different loadings if need be) and run the samples next to each other, if possible with a control that you know contains the two species. That should save you a lot of hassle compared to 2-D gels.
Good luck,
Pierre
Hi Mervi T Hyvönen, there is one way by which you could differentiate between such protein isoforms is by performing 2D- gel electrophoresis (separates isoforms of proteins based on their molecular weight and isoelectric point (PI)).
Hello Mervi,
Unless your gels are of poor quality, there should be no problem in distinguishing two polypeptides with Mw 51 and 55 kDa by SDS-PAGE analysis. I would use a regular Lämmli gel with 12.5 % polyacrylamide, avoiding to overload samples (ideally no more than 1 µg/band, run several different loadings if need be) and run the samples next to each other, if possible with a control that you know contains the two species. That should save you a lot of hassle compared to 2-D gels.
Good luck,
Pierre
I would add a lane in which you run a mixture of your samples. The difference in molecular weight may not be enough to see differences between adjacent lanes easily by naked. But if you get a double-band with your mixture, you have at least a control that there are different sized molecules present.
You could then analyze the lanes closer from a scan with your compurter.
2-D gels are useful for proteomic analysis more than differentiationg two different isoforms. Have you ever thought about capillary electrophoresis? https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_(Analytical_Chemistry)/Instrumental_Analysis/Capillary_Electrophoresis
Thank you all for your answers. I will first try with 1D PAGE, and if not working, then with 2D.. If my antibody is not very specific, then other problems may arise with 2D.
Would it be better to run on 18% gel for longer time, does it separate better than ordinary 12% gel?
A shallow gradient gel (say, 10-15% T) results in narrower bands and hence better resolution.
Alternatively, you could use a transverse gradient to identify the %T for optimal resolution (doi:10.1016/0003-2697(88)90581-7).
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