I worked on Bacillus licheniformis extracellular protease, precipitation was carried out by 95% ethanol, precipitates was dissolved in 0.1M Tris -HCl buffer, sample was desalted by using G-25 beads, that was followed by loading a sample on cation exchange chromatography column, which was equilibrated by 0.5M NaCl Tris-HCl, the protease was collected in unbound fractions, and the protease also did not bind to DEAE Cellulose Column, i.e. anion exchanger, the DEAE Column was equilibrated by deinoized water.
What is the possible solution?
did you try it by using equilibration buffer without NaCl?
Your NaCl is too high. I suggest to use 0-10 mM of NaCl in your Buffer A (equil. buffer column) as suggested by Mahendra..
I agree with Mr Mahendra and Khomaini, several method are ever i read, said that buffer NaCl used with scale micron or mM. Good Luck
You can start DEAE column without NaCl ,and then elute with a gradient 0-0.5 M NaCl
yes Mahendra i load the the sample on DEAE which was not equilibrated by NaCl, Thanks for all,
Please load the sample into the DEAE column after equilibated the column with Tis-HCl buffer without NaCl 2 column volumn. Thank you very much.
different cut off membranes available in the millipore merk
i thing u can use millipore membrane filters (Amicons Ultra centrifugal filters; MWCO-10 kDa; Millepore Ireland Ltd, Ireland) to desalt the enzyme and then u load it in to the ion exchange and gel filtration columns
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