I am trying to get a protein complex (one is around 110kDa and the other is around 25kDa) for Cryogenic electron microscopy (cryo-EM). The main method I am using is to run the sample through a gel filtration. However, I couldn't get the complex. The buffer condition I used is 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT. Is there anything I can optimize to increase the interaction between the two proteins. Thank you!
Gel filtration chromatography is not an equilibrium procedure. If the affinity of the proteins in the complex is not high enough at the concentration applied to the column, the proteins will separate as they become diluted while passing through the column. If you start with a much higher protein concentration, you may get some of the complex to remain together through the column.
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