a problem in protein purification

D. Hodges dh124 at cus.cam.ac.uk
Mon Nov 7 03:54:00 EST 1994

yuling luo (yluo at POBOX.UPENN.EDU) wrote:
: Hi there,

: I am trying to purify a myc-tagged recombinant protein (pI=7.6, 100 kd) 
: from the supernatant of baculovirus-infected insect cells. I first 
: enriched the protein on a cation exchange column, which worked out fine. 
: I then loaded the eluate into an anti-myc monoclonal affinity column and 
: eluted the column with 0.1 M glycine pH 2.5. I found that most of the 
: myc-tagged protein bound to the affinity column. But the eluted protein 
: is in precipitated form. It become obvious when I netralize the elute to 
: netral pH with Tris buffer. Then I would just see a big white precipitate 
: forming. I also tried high pH elution buffer. The eluted protein is still 
: precipitated. My questions are 1) could this be the problem of the 
: affinity column itself? (if it is, I could try different ways to purify 
: it) 2) Could this be the protein's problem? (ie, the protein will 
: aggregate in the absence of other proteins, if this is the case, I am 
: hopeless. 3) anyone out there has similar experience? 

: Your advice is much appreciated.     yl

I've eluted proteins from a monoclonal affinity column using 3M sodium 
iodide (pH7.2) - fractions go staight into dialysis against a more protein
friendly buffer as soon as they come off the column. It's worked very 
for a small protein (30K) but you never know how anything else would
behave - might be worth a try though.

        Debbie Hodges  dh124 at cus.cam.ac.uk  Fax 0223 217838
Rheumatology Research Unit, Addenbrookes Hospital, Hills Road, Cambridge, UK.
	I never could get the hang of Thursdays   (A.Dent).

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