Hi Jothi,
You can make the blue native gel by yourself. It is quite cheap and easy to
make. Other than the standard native PAGE supplies and some buffering
reagents (if you want pH~7, imidazole will do), what you need in addition is
commassie G-250. Please note, that commassie R-250, which you may find in
your lab's protein gel stains, will not work for blue native gel (at lease
didn't work for me). The reason probably is that R-250 has ethyl groups on
the dye, making it more prone to irreversibly aggregate proteins.G-250 has
methyl groups at the same places, so its complex with proteins are not so
bad.
There are some protocols on internet, also nature protocols has an article
for blue native PAGE:
http://www.nature.com/nprot/journal/v1/n1/full/nprot.2006.62.html
In addition to these protocols, here are some of my suggestions:
First, it is not always necessary to make gradient gels. 8~12% PAGE gels
will usually work for regular sized proteins (20~200KD). Also it is not
necessary to make discontinuous gels - meaning you can take the staking gel
out. You might get a silightly broader band, but a continuous gel system (no
staking gel, and the upper running buffer and loading buffer has same pH as
the gel) is easier to design and you do not need to worry about artifacts
caused by the compression effect.
Another thing is, in some of the protocols, the authors suggested using
water to make the G-250 stock solution. But you may find that this G-250
stock is actually a very poor suspension - so the final concentration in the
upper buffer may never be what it is supposed to be. My solution is to make
0.5% EtOH or DMSO solutions of G-250, and add it 1:50 to the upper buffer to
make 0.01% working concentration. The resulting concentration of alcohol or
DMSO should not be high enough to harm most proteins.
Finally, You may try using those SDS-PAGE protein ladders for the BN-PAGE as
size marker. As to my experience, most bands of the fermentas PageRuler will
run to approximately the same position as native proteins of the same size.
Zhijie
----- Original Message -----
From: "KN" <kn...@auckland.ac.nz>
To: <CCP4BB@JISCMAIL.AC.UK>
Sent: Thursday, February 12, 2009 10:30 PM
Subject: [ccp4bb] native gel
Dear all,
I am wondering if anyone is working on Blue Natve PAGE or other alternate
methods. I need some help to troubleshoot my Native PAGE experiments. It
will be great if anyone can help me in this regard. I am working on
membrane
proteins which have pI around less than 7. i need to run Native PAGE for
my
samples which are purified with nonionic detergents. When i tried to run
inhouse 4-16% gradient Native gel at running buffer pH of 8.9, the samples
are getting stuck at the wells itself. I just found out that invitrogen
Blue
Natvie PAGE may be a good option but due to the cost if anyone can suggest
me some other alternate methods, I will really appreciate it.
Thanks in advance,
regards,
Jothi,
