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Protein won't immobilize on CM5 chip

  • myouse
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7 months 3 weeks ago - 7 months 3 weeks ago #1 by myouse
Protein won't immobilize on CM5 chip was created by myouse
Hi all, I'm running SPR studies on an enzyme found in bacteria and in humans using a CM5 chip. The bacterial enzyme immobilizes well and results in high RU (first picture). However, the human enzyme will not immobilize to the chip (second picture). In both cases, the running buffer is PBS with 2% DMSO. The proteins are prepared as 50 ug/mL solutions in 10 mM sodium acetate, with slightly different pHs. These conditions are exactly what has been used in the literature for the same protein, yet I haven't been able to see any immobilization. Has anyone ever seen a similar sensorgram before? Does anyone have any ideas for solutions? Thanks in advance!
 
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Last edit: 7 months 3 weeks ago by myouse.
  • Arnoud
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7 months 3 weeks ago - 7 months 3 weeks ago #2 by Arnoud
Replied by Arnoud on topic Protein won't immobilize on CM5 chip
Hi,

Looking at the second sensorgram there is no pre-concentration ( www.sprpages.nl/immobilization/ligand-pre-concentration ). There are some possibilities why not: 1) wrong pH of immobilization buffer ; 2) too low ligand concentration (check stock concentration); 3) the dilution of the stock into the immobilization buffer is altering the pH too much; 4) ligand stock conrains TRIS or other compunds with primary amines..

Kind regards
Arnoud
Last edit: 7 months 3 weeks ago by Arnoud.
  • myouse
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7 months 3 weeks ago #3 by myouse
Replied by myouse on topic Protein won't immobilize on CM5 chip
Yes!! The ligand was ordered from Sigma and was in buffer containing tris. Such an easy fix (and an oversight on my part...), so I'm ordering lyophilized protein now. Thanks so much!
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