[Protein-analysis] Re: 1. Problem with sephadex G 75 (Catherine Wong)

Clement Angkawidjaja via proteins%40net.bio.net (by clement from bio.mls.eng.osaka-u.ac.jp)
Mon Jun 25 19:49:16 EST 2007


Re:  1. Problem with sephadex G 75 (Catherine Wong)

Hi Catherine,

My GF buffer which i solubilize my protein
> 0.05M Tris CL
> 0.05M Nacl
> 0.02% sodium azide
> pH 8.0
>
> is there a problem with this buffer??
> is the Nacl too low??
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.

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?

> 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.

Hope this helps.

clement


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To: <proteins from magpie.bio.indiana.edu>
Sent: Tuesday, June 26, 2007 2:01 AM
Subject: Proteins Digest, Vol 25, Issue 5


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> Date: Sun, 24 Jun 2007 09:35:33 -0700 (PDT)
> From: Catherine Wong <catwfy from yahoo.com>
> Subject: [Protein-analysis] Problem with sephadex G 75
> To: proteins from magpie.bio.indiana.edu
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>
> My GF buffer which i solubilize my protein
> 0.05M Tris CL
> 0.05M Nacl
> 0.02% sodium azide
> pH 8.0
>
> is there a problem with this buffer??
> is the Nacl too low??
>
> There's suggestion of using higher Ionic strength:
> 0.010 M tris
> 0.8M nacl
> 0.1mM EDTA
> 7 mM p-ME
> 0.01% triton-x
> pH 7.5
>
> or should i change to this??
>
> pI of my protein is  6.99 theoretically.
> I run my column with my GF buffer at 1ml/min
> bed height 27cm..cross area 0.79
> bed volume would be ~23 ml.
> Loaded 1 ml of 12 mg/ml BSA (just to see the packing)
> result :
> I don't see a peak at all.
> what is wrong???
>
> 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??
>
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