We want to purify a 88 kDa bacterial protein from extracellular crude extract through gel permeation/size exclusion chromatography. I'm just beginner in this field and wonder about the basic parameters such as:
type of resin
column height and diameter
mobile buffer (strength and pH)
sample volume
flow rate
each fraction volume etc.
Kindly help me.
buffer here you can use either tris or phosphate . pH should be nearer to your protein PI. FRate depends on your resin volume and binding capacity. Sample volume depends on what was the volume of resin you are taking and its binding capacity.
Try to minimise flow rate and volume of sample for better purity.
I had the same questions in my mind a few weeks back, go through the GE size exclusion chromatography manual, it details everything.
http://proteins.gelifesciences.com/~/media/protein-purification-ib/documents/handbooks/size-exclusion-chromatography-handbook.pdf?la=en
Dear Sandeep:
Which kind of protein are you interested in? Parameters to be used vary on the basis of the protein to be purified. Gel filtration resins such as Sephacryl or Sephadex work fine. This kind of columns should be long and with small diameter. Sample volumen depends on you protein concentration and the resin.
Good luck,
Thank you all for valuable suggestions, the protein is a secreted bacterial toxin of 88 kDa, having a signalling sequence and a hydrophobic domain. The theoretical PI of protein is 9.
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