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[Mark_lab]

hi everybody,
i'd like to perform this assay in my lab, we have gotten a protocol by internet and i've tried it some times, but i've some doubts.

in this protocol don't mention the amount of protein extract that i have to incubate with WGA, only mention a volume (0,5ml). i don't know if exists a critical amount (minimal or maximal)

in the wash steps we should use a buffer with PBS1X/TritonX100 0,1% but in some papers i've seen that this wash buffer includes inhibitor proteases; should be included or is not important?

finally, in the elution step i don't know if i could use directly SDS-loading buffer to elute the  glycoproteins...

I probed this protocol some times and in all we have recovered a poor amount of protein, and if i perform a western with this samples it's almost impossible to detect something.

if someone could help with these doubts or know about another protocol, i'd be greatful
thanks so much!

[biored]

Hi Mark,

I am using the protocol of this reference:

J Biol Chem. 1988 Dec 15;263(35):18911-9.
Isolation and characterization of human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. Major sialoglycoproteins carrying polylactosaminoglycan.
Carlsson SR, Roth J, Piller F, Fukuda M.
The column is packed in a 5 mL pipette (WGA-Sepharose beads, GEHealthcare, 4-5mL of beads, column needs to be long and thin). I start with 0.9g isolated glomeruli and follow this protocol almost exactly ending with a total volume of 6mL of protein soup. The difference are that I never ultracentrifuge my sample but spin it only at 2000g to remove big debris, I use a more powerful protease inhibitor cocktail, I don't check the buffer flow.
All buffers are cold. I do the whole procedure on my bench. Rather easy, probably not totally clean but...
I enrich highly for most heavily N-glycosylated and O-glycosylated proteins as confirmed by Western blot and MassSpec.

Edited by biored, 25 April 2012 - 08:22 AM.