I am trying to develop a heparin affinity column method using a 100mM to 1M NaCl step elution. My protein of interest actually elutes at a 0.5M NaCl step, but though it elutes in a nice, sharp peak, it is not completely eluted: I get peaks in subsequent blank runs. I am using the lower end of recommended pH for mobile phases so all I can think of to get more complete elution is including a nonionic detergent like NP40 or Tween or CHAPS in my elution buffer. Alternate strategies or detergent suggestions are most welcome.
John Gallagher, Iduron Co. and University of Manchester, UK
How much protein of interest remains on the column? 100% yield is difficult to achieve; a non-ionic detergent may help but then you have to remove the detergent and may incur losses in doing so. We (www.Iduron.co.uk) sell a heparin affinity column and I can send you a sample free of charge if you'd like to try it.
How do you regenerate your column - this procedure should remove most if not all proteins that remain attached to the heparin matrix and subsequent blank runs should then be substantially free of protein
We also wash the column with 2 M NaCl between uses. The first use of the column frequently provides lower yields than subsequent runs as if there are some very high affinity sites that need to be saturated.
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