I have been trying to purify glycoroteins but they are fail to appear on SDS PAGE because they are highly coated with sugars. How can i Stain them through manual method?
I work with glycoproteins and I have to agree with Manchala, the bands will appear as a smear. I have attached an image of what my protein looks like from a SEC column after His purification and stained with coomassie. This smear may be due to the alternative glycosylation that can occur when it is produced. I would suggest doing a PNGase assay in order to look at the protein without the glycosylation. This would show a single band for the PNGase (around 36 kDa) and your protein of interest without glycosylation. If there is a single band then you know you have what you want.
I hope this helps.
Please have a look at this kit:
http://www.piercenet.com/product/glycoprotein-staining-kit
(I have not used it so I have no first-hand experience if it works well.)
Best regards, Christian
Try silver staining, much better than coomassie. The worst you can get is negative staining (clear spot surrounded by high background).
In SDS PAGE glycoproteins appears in a smear, so you can not see as a sharp bands. you can go for a gradient gel electrophoresis.
all the best
I work with glycoproteins and I have to agree with Manchala, the bands will appear as a smear. I have attached an image of what my protein looks like from a SEC column after His purification and stained with coomassie. This smear may be due to the alternative glycosylation that can occur when it is produced. I would suggest doing a PNGase assay in order to look at the protein without the glycosylation. This would show a single band for the PNGase (around 36 kDa) and your protein of interest without glycosylation. If there is a single band then you know you have what you want.
I hope this helps.
Yes, i got the same smear on my gel. I will try this PNGase assay because due to these sugars some glycoproteins are not bind to ion exchange column.
Dear Bilal,
Glycoproteins can be resolved and visualized in SDS PAGE using the standard Laemmli et al, but these glycoproteins as mentioned earlier appear as smear. They also show poor staining pattern with coomassie staining... Try PAS (Periodic acid–Schiff) staining... this is specific for glycoproteins... Also you can try lectin blotting as well...
If you paln to deglycosylate with PNGase... use PNGase A for plant/insect glycoproteins... PNGase F doesnot work in certain cases (in the presence of alpha1-3 fucose)
If you are trying to partially purify or fractionate the glycoprotein, I would recommend Lectin sepharose for the same... Ion exchange at times doesnot bind glycoproteins effieciently...
All the best for your efforts
Saravanan K
Hi, I just tried the Pierce glycoprotein staining kit, and no bands showed up except for two strong bands for the positive control provided by the kit. I loaded 10ug proteins and added DTT to the samples before denaturation at 70C for 10 minutes...would DTT + heating be a problem?
Alice, would you be so kind to tell me how you usually prepare your protein samples?
Thanks so much!
--sunnie
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