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

Catherine Wong via proteins%40net.bio.net (by catwfy from yahoo.com)
Wed Jun 27 11:42:44 EST 2007


Hello everyone
thanks for replying.


Dr Engelbert Buxbaum
>>That all largely depends on the protein you want to separate. For  
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


>>The first thing to make sure is that your column is properly packed.  
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.
>>The next step is to calibrate the column with a couple of marker proteins,  
suitable kits are commercially available from the usual suspects.

>>It should not normally be necessary to sanitise the column, if you filter  
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 
>>I do not think NaCl is too low. pH may be the problem. You mentioned your 
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.

>>HOwever, you could not see any peaks of BSA (or could you?), that's kinda 
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 

> then : i clean the column with 0.5M Naoh
> and GF buffer
>
> BUT>>>> after a volume of GF buffer pass through... the buffer can't seem 
> to pass through .. i tried and waited.
>
> is it because of the 0.5M naoh??
> should i wash with NACL instead??

>>You should first clean the column with milliQ water after the buffer, then 
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

       
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