[Protein-analysis] Problem with sephadex G 75

Catherine Wong via proteins%40net.bio.net (by catwfy from yahoo.com)
Sun Jun 24 11:35:33 EST 2007


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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