purification problem. - most of my protein is washed away.... (Apr/03/2006 )

i have two great problems in purifying my protein by sepharose beads (GST)....at last i have successfully dissolved my insoluble protein but now i am having problems with attaching it to the beads.... ....first problem is that only very little of my protein seems to attach to the beads....have tried old and newly received beads but same results... second problem is that during the first wash half of the protein is washed away ...please can you help me to troubleshoot this....thank you ver much...have a great day all!

what did you do to solubilize it? imidizole, urea, NaCl?

have you tried a solubility gradient? does this make sense...make changes to your buffers to increase solubility, but in small increments; perhaps this would allow you to find a happy medium between insolubilty and oversolubility

at an appropriate pH?

aimikins, do you remember protocol i have posted something about "probe and pellet"...i have used it and it went very good....dissolved in sarkosyl vortex for 5 min......i still have around half of my protein in the insoluble portion though, but anyway what ive got in the soluble portion is just enough for me....
but what you mean is that now my protein is over soluble?? thats why it is not attaching to the beads?? please can you clarify this further, thank you very much again.

Oh, geez, Kathy...I hope I can be helpful here.

I don't know much about sarkosyl as a solubilizing agent, but if it works like many solubilizing agents in protein purifcation...hmm...let's give an example.

If I have tagged-protein X that I am trying to purify via a column method...I find that it is insoluble and so I add, say, 2 M NaCl to my lysis/wash and elution buffers to increase solubility throughout the protocol. I then find that all my protein is being washed out, and not binding effectively to the column. OK, then, next I could try 1 M NaCl...or better yet, a step-gradient of NaCl...the solubility will be dependent at least somewhat on the concentration of solubilizing agent, right?

so, I would add wash buffer with increasing amounts of the solubilizing factor, retaining all fractions until I figured out the threshhold which would increase solubility but keep the protein from washing away. I would do this with a pretty small batch. When I determined the concentration of solubilizing agent which left most of the protein on the column during washing, I would wash with that buffer...then elute at a slightly higher concentration of solubilizing reagent.

Does this make sense? I hope it applies to your system...does anyone else have a better way to explain this?

aimkins thanx a lot for the information!. I dont know how much I can change the composition of the elution buffer. And also I dont want to elute. i am ok with protein just being attached to the beads....i want to use it like that. But your point of finding an optimal concentration of solubilizing agent is so true i guess. I have to work on that. ...it seems i have millions of problems with this purification. and on the top of all degradation!

ah, you're doing OK. you've been working on this, what, a few weeks? it takes time...don't worry. you just have to find the right ionic conditions for your protein and sometimes it takes troubleshooting

good luck!!!

Hi everybody,

I´ve been told that expressing your construct at low temp (ie, 16-18º) o/n sometimes yields a higher amount of soluble protein. Has anybody tried something about it?
I´m having lots of problems with an insoluble GST fussion: after solubilizing with Sarkosyl, eluting from the column and dyalizing extensively, I can´t rebind the protein to the beads!!

miguelon

miquelon, i never heard of inducing at such a low temperature.....but maybe its worth trying...
what do you mean that after sarkosyl you run it through the column? i add triton to sequester sarkosyl but maybe its not enough.... ....very little is attaching to the beads here also.....

Hi Kathy.

Most of my construct remained in the insoluble pellet. I was able to solubilize it and to bind it to my column in 1% Sarkosyl + Triethanolamine. When I first tried to elute it in mild conditions, most of it remained attached to the column, so I eluted it adding sarkosyl to the elution buffer. I dialyzed it extensively, but I was not able to rebind my protein to new gluthathione-beads for my experiments.

I´ve been told recently that expressing the protein very slowly, you may obtain a higher proportion of soluble protein... I will try and keep you updated.

Miguelon

miquelon, i never heard of inducing at such a low temperature.....but maybe its worth trying...
what do you mean that after sarkosyl you run it through the column? i add triton to sequester sarkosyl but maybe its not enough.... ....very little is attaching to the beads here also.....