Hello,
I am trying to find a buffer composition that contains low or no NaCl for a protein that is purified in 500mM NaCl. The reason for lowering the salt is trying to obtain good vitreous ice for cryo-EM. When I attempt to drop the salt it causes aggregation. I have previously tried using a phosphate buffer which does stabilise the protein but it still produces really grainy ice and my protein is fairly small which makes seeing particles really difficult. I also tried sodium thiocyanate which gives beautiful ice but (not surprisingly) kills enzymatic activity by the protein. My current approach is trying non detergent sulfobetaines but if anyone has any suggestions of things I could try I would be very grateful!
Hi,
Please reduce the NaCl concentration (to 50 or 100 mM) in your sample by desalting. You may use 100 mM Tris or phosphate buffer, pH 7.5 containing 50 mM Nacl (final concentration) for cryo-TEM studies. This buffer can be directly used for buffer exchange while desalting.
Hope this helps.
Anoop
Hi,
You can also add small amount of L-Arg (50mM) during purification to prevent aggregation when you lower down the salt concentration.
Good Luck
Sunil
Even you can use sugar stabilizer like Sucrose, Mannitol or Sorbitol for stability of protein.
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