glycoprotein staining
Rob Hobson
a simple method to non-specifically recognise glycosylated proteins directly
on the gel, I have read about staining techniqures such as alcian blue, but
I'm not sure where to procede.
vetim...@hau.nic.in
I am giving you few references for glycoprotein staining on SDS-gels:
a)Periodoc acid -schiff (PAS) method:
Fix gel in 12.5% TCA for 30 min.
Rinse in H2O
Place in 1% Periodic acid in 3% acetic acid for 50 min.
Wash in several changes of H2O
Transer to fuchsin-sulphite solution in the dark for 50 min
(Procedure to make fuchsin-sulphite solution-make a soln of 16 g
potassium metabisulphate and 21 ml of concentrated HCl.Add 8 g of basic
fuchsin and stir gently for 2 h at room temp.Then add acid washed
charcol and after 15 min filter{stored cold,it works for several months}
Finally,place the gel in 0.5% potassium metabisulphite(3 times for 10
min each change of 30 ml per gel).
Wash in H20 until bands become visible
Take care, do not wash too much other wise bands will disappear
Take photos immediately.
store in 7% acetic acid
**There is one another variant of this method which I followed for
staining a gP of Schistosoma sp.It is as follows-
Immerse gel in 12.5% TCA - 30 min
Rinse in water-0.25 min
Immerse in 1%Periodic acid in 3% acetic acid-50 min
Washed thoroughly in water-Overnight
Transfer into fuchsin-sulphite stain in dark-50 min.
(Procedure to make fuchsin-sulphite stain -dissolve 2 g of basic
fuchsin in 400 ml of H2O with warming the solution and then cool it.
Filter.Add 10 ml of 2N HCl and 4g of K2S2O5 in it.Place the solution in
a stoppered bottle and kept cool overnight.Next day, stir 1 g of
activated charcol ,filter and add sufficient 2N HCl(10 ml or more)until
a drop dried on a galss slide DID NOT turn red. Stored stoppered in a
cool dark place)
Wash the gel with 3 changes(10 min each)of 0.5% Na2S2O5-30 min
Wash in frequent changes of water until excess stain was removed.
Take care, do not wash too much other wise bands will disappear
Take photos immediately with in 5 min.
store in 7% acetic acid
b)Peroxidase -conjugated lectins
Olden and Yamada,1977, Anal.Biochem.,78,483-490.
c)The protein protocols handbook by J M Walker,Page no.627(Staining of
glycoproteins on SDS gels
Good luck,
Devender Kr. Saini,
Research associate,
Immunodiagnosis Lab,
Dept. Veterinary Parasitology,
College of Veterinary Science,
CCS Haryana Agricultural University,Hisar,Haryana,
India-125004.
----- Original Message -----
From: Rob Hobson <robh...@yahoo.com>
Date: Wednesday, May 22, 2002 2:49 am
Subject: glycoprotein staining
> I am interseted in identifying glycosylated proteins by SDS-PAGE.
> Is there
> a simple method to non-specifically recognise glycosylated
> proteins directly
> on the gel, I have read about staining techniqures such as alcian
> blue, but
> I'm not sure where to procede.
>
>
>
>
> <" target="l">http://www.biowww.net/forum/read.php?f=6&i=1018&t=1018>
>
>
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