I carried out a deglycosylation test to see if there will be difference in the electrophoretic mobility of C57BL/6 infected-ME7 and SShpPrP Tg ( transgenic mice expressing sheep prion protein) infected-ME7 scrapie strain using PNGase F (P0704S) from Bio lab. Same rough band was observed for incubating samples with PNGase F for 1 hour and 24 hours. Also, l got same result when l carried out the electrophoresis at 100v, 90v and 85v. Samples were denatured by heating at 99 degrees for 15 mins prior to loading which has been my protocol for immunoblotting involving C57BL/6 and SShpPrP Tg mice infected ME7 scrapie strains.
I will be glad if any one can help with suggestions towards getting a neat un-glysosylated band after deglycosylation.
You mean the wavy bands in the deglycosylated samples from transgenic mice? It looks to me as if there is some precipitation/heterogeneity in those samples. Try centrifuging the samples and analyzing whatever is left in the supernatants (or including urea in the sample buffer).
Hope it helps!
firstly check what size PNFase F is to dismiss contamination from enzyme (bands look to small for this). You may also have degraded product present due to treatment. Are your controls set up under the same conditions with - PNGaseF? Not sure whether your samples are denatured or not for PNGase treatment? if denatured then unlikely to be protease activity.
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