Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protein Science > Protein Forum
Register Search Today's Posts Mark Forums Read

Protein Forum Protein Forum


[Protein-analysis] Re: Problem with sephadex G 75

[Protein-analysis] Re: Problem with sephadex G 75 - Protein Forum

[Protein-analysis] Re: Problem with sephadex G 75 - Protein Forum


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 06-27-2007, 04:42 PM
Catherine Wong
Guest
 
Posts: n/a
Default [Protein-analysis] Re: Problem with sephadex G 75




Hello everyone
thanks for replying.


Dr Engelbert Buxbaum
extracellular proteins I'd use something like 100 mM NaCl, 50 mM Tris. For
intracellular proteins a potassium-based solution is better, possibly with
some Mg too (but which may also activate proteolysis). Also note that the
cytosolic environment is reducing, some DTT or bME may help

My protein is intracellular protein(cytosol).
But I'm trying to avoid DTT or p-ME until the last step which is IEC.
Do you think it's better to add it??
So far my protein is good as far as it's keep in -80.

I'm actually using BSA to make sure my column is properly packed.
I don't have the fund to buy that at all even though I know i'm suppose to use these cheap alternatives
Anyway, I did SDS page and found out that my BSA starts to elute at fraction 13-25.
However, the fractions mostly are together with unspecific bigger bands.
I'm not sure why is this happening. Any idea?
Not only that.. I'm confused ..
Why is my BSA still eluting /separating at fraction 25..since theoretically my bed volume is about 22ml.
each fraction was 1 ml


Simply inject a sample of Dextran blue and 1 mM ATP, which should give
nice peaks at the excluded and total volume respectively, symetrical and
not too wide. The dextran blue you can see marching down the column, the
band should be sharp, without tatters and horizontal.
suitable kits are commercially available from the usual suspects.

your samples through a 0.2 um low-protein binding membrane. A brief spin
in an Eppendorf-centrifuge can achieve the same, but be careful not to
disturb the pellet.
I use a 0.2 um filter that is used for normal filter of antiobiotic. I'm assuming it's ok.
I think it still bind my protein but not that much as I run SDS page to look at it.
basically i just don't have budget at all for that as well.


Clement Angkawidjaja
protein's theoritical pI is about 6.99. pH 8.0 may be too close to the pI.
I'd suggest increasing the pH by 1 (or decrease to 4.5 or 5, depending on
which one your protein is most stable). Remember that the pI of BSA is
around 5 should you use the same buffer for BSA.

Oh yes.. I forgot that BSA pH is around 5.. I'm actually using pH 8 (this is my protein's GF buffer)
Will that affect the GF?
I'm using BSA as a standard to make sure everything is packed properly.

weird. How much volume of buffer did you use to elute your BSA?

I did see a peak if I take the lowest reading as the minimum value ..
Therefore, I do get a peak at fraction 13-22 by A280 nm
However by SDS page, I see fractions with unspecific (bigger) bands in fractions 13-25.

I'm guessing initially my fraction shouldn't have any bands after the bed volume of ~22 ml..
but at fraction 25(25ml) there still has.
I did 2x volume of buffer to the bed volume = ~52 ml


with NaOH. Repeat with water after NaOH prior to buffer. Tris solubility
changes as pH changes. Furthermore if you include some cations in your
buffer, they may crystallize when they meet NaOH.

I think the safest bet is to use NaCl to wash the column.

thx .



Agape,
Catherine Wong
Dept of Genetics,
Institute of Biological Sciences,
Faculty of Science,
University of Malaya,
50603 Kuala Lumpur.
Tel : 03-79675994/5851
Fax : 03-79675908


---------------------------------
Building a website is a piece of cake.
Yahoo! Small Business gives you all the tools to get online.
Reply With Quote
Reply

Tags
problem , proteinanalysis , sephadex


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
plasmid re-ligation problem Vagelis Protocols and Methods Forum 4 06-20-2012 08:29 PM
2D gel running problem cosmis85 Proteomics Forum 4 12-31-2009 05:26 AM
SDS-PAGE problem Steve Nothwehr Protocols and Methods Forum 2 10-16-2009 08:59 AM
2D gel running problem cosmis85 Protocols and Methods Forum 0 02-21-2007 10:08 AM
FW: SDS-PAGE problem Deanne Bell Protocols and Methods Forum 0 03-05-2004 05:27 PM


All times are GMT. The time now is 06:33 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.11852 seconds with 16 queries