Hi there,
I plan on doing IEC tomorrow and need to dialyze my protein to keep a negative charge...the plan is to dialyze in Tris pH 8 (pi of my protein is 6) does that sound right...I don't have to add NaCl to the buffer right (NaCl is what I'm eluting with)..I seem to have written in my book "add NaCl to Tris and dialzye O/N" but I'm pretty sure it's wrong..
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#2
K.B.
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Posted 02 November 2011 - 12:05 AM
Your pH is OK.
Some proteins require salt or they will precipitate.
Amount of acceptable salt in sample (and mobile phase A) would depend on at what salt concentration it actually elutes during IEC. I.e. if your protein elutes early, you should keep salt concentration as low as possible; if it elutes late you may increase salt concentration in sample and mobile phase A.
Some proteins require salt or they will precipitate.
Amount of acceptable salt in sample (and mobile phase A) would depend on at what salt concentration it actually elutes during IEC. I.e. if your protein elutes early, you should keep salt concentration as low as possible; if it elutes late you may increase salt concentration in sample and mobile phase A.
#3
mdfenko
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Posted 03 November 2011 - 06:09 AM
salt during dialysis also helps to reduce non-specific binding to the membrane.
as k.b. was saying, you can add salt up to the point that it may interfere with your protein of interest's binding to the matrix.
as k.b. was saying, you can add salt up to the point that it may interfere with your protein of interest's binding to the matrix.
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