Dear all,
I'm trying to stain sugar groups attached to my protein with Glycoprotein staining (Thermo Scientific Pierce Glycoprotein Staining Kit #24562) after gel electrophoresis.
I've added 4 ug/well and 2ug/well of antigen samples (with milliRo and 4x laemni and 2-mercaptoethanol), which would be sufficient according to the manufacturer.
Only the positive control (HRP) seems to give magenta bands.
Thanks a lot!
Hi Liz,
I recommend you to perform a SDS-PAGE of your sample. If you already have done it, once both positive and negative controls are ok, i guess that your sample isn't a glycoprotein, or the glycoprotein was not glycosylated.
How do you performed the purification? Is this a recombinant glycoprotein/protein? If yes, are you using a m.o. that makes this post-translational modification? (unfortunally, I don't know what AWA and ES means).
As your positive control is working properly, seems that the protocol is OK.
- You should try to increase the concentration of sample.
- You can try to increase the incubation time in periodic acid (oxidation reagent) just to make sure, your glycosylated proteins (antigen) are all oxidized. If the sample has low concentration of glycosylated protein, it will need a little longer time for oxidation process. Also make sure the gel is incubated in dark during staining.
- Thermo Scientific Pierce Glycoprotein Staining Kit works good according to my experience.
- There is also a manual protocol without using kit if you would like to.. I have also used this reference protocol and it worked good for me.
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