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Protein Glycosylation and SDS-PAGE


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#1 SusieQ

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Posted 14 May 2014 - 11:54 PM

Dear all,

 

My labgroup has been trying out the Pierce Glycoprotein Staining Kit (http://www.piercenet...in-staining-kit) to detect glycosylated proteins on SDS-PAGE. However, the first time we tried it, there was not a single glycosylated protein visible on the gel, even though the manufracturer's protocol was followed and the positive control gave the expected result. The samples we used were membrane proteins isolated from different cell lines (CHO and PC12), and a conventional SDS-PAGE procedure was done. We highly doubt that there are no glycosylated proteins present in these lysates and it got us wondering whether the sample prepatation procedure can have an effect on protein glycosylation?



#2 jerryshelly1

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Posted 19 May 2014 - 05:21 AM

How are you extracting your protein



#3 SusieQ

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Posted 19 May 2014 - 10:01 PM

We lysed cultured cells using RIPA buffer (Sigma #R0278) supplemented with protease inhibitors (prepared freshly). So basically,cells were cultured in 6-wells plates, first washed with PBS and then protein was extracted by adding RIPA buffer + protease inhibitor cocktail (Roche, 1 tablet per 10 ml RIPA buffer), 150 ul per well. Samples were kept on ice afterwards and mixed with 4x Laemmli Buffer before loading onto a Criterion Pre-cast Gradient gel (4% - 15%).

R0278
R0278





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