I am looking for a method to detect several glycosylated proteins.
Any one have a good Idea?
I found this good product from Invitrogen,http://probes.invitrogen.com/lit/catalog/1/sections/1143.html
It works great.
Your link doesnt work. What was the method and product to detect glycosylated proteins?
Also what types of glycosylated proteins? N-glycosylated? O-glycosylated? proteoglycans? there are tons of detection methods for the different types of glycan chains that can be added to proteins?
Can you give some more information on the cell type and organism you are working with?
note - topic moved to post-translational modification forum
Pro Q Emerald is a non specific glycosilated protein detection stain. It reacts with periodate-oxidized carbohydrate groups.
So if you in need of a non specific fast on gel staining this is a great tool to use. I used it couple of times with a combination of Sypro Ruby and got good results which are could be measured.
Here is the link to the product.http://probes.invitrogen.com/media/pis/mp21855.pdf
I know that Invitrogen have some products that can detect the O-GlcNAc did not try it.
Here is the link.http://probes.invitrogen.com/media/pis/x33365.pdf
Interesting. I guess this would be useful for detecting all oxidizable glycans added to proteins. Im wondering what the applications might be? What question were you specifically asking?
How useful is it to show that X % of proteins are glycanated per se?
As a card carrying glycobiologist Im mostly curious? How does this information help you in your experimental hypothesis?
I mean all secreted proteins contain glycans by definition otherwise they would not pass through the ER - golgi and be secreted. This seems to be a major point missed by most graduate school curriculums!
The main question that I asked was regarding the amount of glycosylated proteins on the Lysosomal membrane.
I did 2D-IEF and used it to see the over all amount of proteins which were ~350. Then I wanted to know how aging and Alzheimer's effected the number of glycosylated proteins in that fraction.
So for an over all count it was great.
As you know some many diseases effect post-translational modification of protein i.e. phosporylation glycosylation some say also pegylation.
In any case I found that there are changes between young and old rat liver fractions (not published yet).
Hey Guy and Rusty,
I really feel that differential lectin staining is the way to go. Different lectins (Con A, PNA . . .) will specifically bind to different glycosylation modifications (Branched alpha mannose, Gal (betaB1,3) GalNAc) and act as reporters. There are some distinct advantages to this technique. Lectin staining can be used in immunoprecipitation, flow cytometry, and even immunohistochemistry. The lectins can be variably modified (Biotinylated, fluorescently tagged, HRP tagged) depending on the application. Differential lectin staining can reveal a large number of specific glycans. This is a very useful technique.
I attached a paper that uses lectin staining for histochemistry in a model of neuropathology (should be what you are looking for, Guy). The paper details the use of 11 different lectins, and there is a table that lists the specific glycans (table 1).
One other point, there are many chemicals that alter cell surface glycosylation (swainsonine, DNJ . . .) to allow for experimental controls for the specificity of lectin binding (one can also compete binding with the relevant sugar).
We routinely use lectin staining in my lab, and I am happy to answer any questions.
Here is another great paper detailing this technique:http://www.hh.um.es/Abstracts/Vol_24/24_1/24_1_1.htm