Hi all I want to purify a protein (Pi:4,0) and I am use a Typical buffer (50mM Tris pH: 7,4, 200mM NaCl and 10mM glucose ( decreased the aggregates and It is better for the crystallization than the glycerol)) I don't know If it's better use a low PI around 5,5-6 or change the NaCl concentration or the salt, like KCl. I know its very complicated to determine the perfect buffer.
In this buffer the protein interactions are low or It forms aggregates because when I spin the protein (13,2kRPM) the oligomers/aggregates in the solution transform in the monomer.
P.D: I sonicate the cells at 5' (total) Pulser: 10s ON 10s Off at 33% Amplitude. Is it correct?
Thank you
Hi David
As a rule for choosing a buffer in ion exchange to use the following:
for Anion exchanger use a buffer 0.5–1.5 pH units greater than the pI of the protein of interest while for Cation exchanger use 0.5–1.5 pH units less than the pI of the protein of interest. Sometimes we don't know PI of the target protein in this case it might be good start with the buffer you've mentioned. You can read more about it in BioRad link
http://www.bio-rad.com/en-eg/applications-technologies/liquid-chromatography-principles/ion-exchange-chromatography
Hope this helps
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