Hi everyone,
I am currently struggling with a recurring issue during my western blot analysis. I'm trying to determine the expression variation of an alternative splicing product of my interest protein, which is approximately 75kDa and 62kDa for the full length and exon skipping isoform, respectively, but I always find, in addition to these bands, strong signals at higher MW. Since this protein is N & O glycosylated and palmitoilated I think that all the bands that run at higher MW are my interest protein post translationally modified rather than nonspecific bands. Is there some way to remove this modifications?
As Adam said you can remove the N-linked glycans with PNGase F. However for efficient removal you will need to denature the protein and reduce and alkylate the disulfide bonds. If you require the native structure this may be an issue. You can use palmitoyl protein thioesterases for removal of the fatty acids. The O-linked sugars may not be possible to remove without degrading the protein if you use the chemical method for removal. There are some enzymes for cleaving O-glycans but the do not cleave all the O-glycan structures (endo-GalNAc-ase D and endoGalNAc-ase A). These enzymes are active only toward the unmodified GalNac's and not sialic acid terminated structures. O-linked oligosaccharides are quite variable in structure and are often extended with additional sugars. However O-links are quite small in size so they may not make much of a difference to the overall size of your protein depending on the number of O-linked sites there are of course.
N-glycosylation can be removed by treatment with the enzyme PNGase F.
As Adam said you can remove the N-linked glycans with PNGase F. However for efficient removal you will need to denature the protein and reduce and alkylate the disulfide bonds. If you require the native structure this may be an issue. You can use palmitoyl protein thioesterases for removal of the fatty acids. The O-linked sugars may not be possible to remove without degrading the protein if you use the chemical method for removal. There are some enzymes for cleaving O-glycans but the do not cleave all the O-glycan structures (endo-GalNAc-ase D and endoGalNAc-ase A). These enzymes are active only toward the unmodified GalNac's and not sialic acid terminated structures. O-linked oligosaccharides are quite variable in structure and are often extended with additional sugars. However O-links are quite small in size so they may not make much of a difference to the overall size of your protein depending on the number of O-linked sites there are of course.
I like the siRNA comparison to non siRNA, very nice ideal.
Thanks
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