9 replies to this topic

[asyura]

hye everyone..
i am quite new in this purification field..
recently, i am trying to purify my histag protein (C-terminus)..my vector is pET52b(+) and my host is BLR (DE3)..
the protein is well-expressed..
but..my problem comes when the protein is very hard to purify using AKTA..
i tried to purify using Ni-NTA spin column (QIAGEN)..and i can get very nice and clear single band..
unfortunately, when i tried to purify using AKTA..my protein yield is very low..until i couldnt see the band clearly when i run by SDS page..
i use HisTrap HP 5ml column by GE

lysis/binding buffer:
7M urea
100mM NaH2PO4
100nM Tris-HCl
pH8

washing buffer:
20mM sodium phosphate
0.5M NaCl
20mM imidazole
pH7.4

elution buffer:
20mM sodium phosphate
0.5M NaCl
500mM imidazole
pH7.4

i did try pH6.5 for elution buffer even the result is better compare to elution buffer pH7.4, but it is still not enough since i need very high concentration protein to inject into mice..

hope everyone can help me..thanks

Edited by asyura, 06 April 2011 - 04:04 AM.

[BioMiha]

did you pass your sample through a membrane before applying it to the column. I lost the whole sample once because it stuck to the membrane.

[asyura]

BioMiha, on 06 April 2011 - 08:11 AM, said:

did you pass your sample through a membrane before applying it to the column. I lost the whole sample once because it stuck to the membrane.

i did filter the sample for degassing..i have no idea if it was a reason of the faint band..

[protolder]

hola, your protein is insoluble if you don´t add urea, I think. why don´t you analize flowthrougt .if  the protein doesn´t bound could be because 6His are hidden, why dont you add some reducer, Nickel columns admit untill 10mM mercapto ethanol; and I think  2mM could improve your yield. Buena suerte

[asyura]

protolder, on 07 April 2011 - 03:51 AM, said:

hola, your protein is insoluble if you don´t add urea, I think. why don´t you analize flowthrougt .if  the protein doesn´t bound could be because 6His are hidden, why dont you add some reducer, Nickel columns admit untill 10mM mercapto ethanol; and I think  2mM could improve your yield. Buena suerte

hye, i did analyze my flowthrough.. (after washing)..but my protein was not there..means it is stuck at the column but doesnt want to elute out once i put the elution buffer..it seems like my protein is too stubborn..bind strong to the column..i will try to put mercaptoethanol..but, i am still wonder what is the purpose of reducer. i read before about it, but i cant understand much..
thanks anyway

[protolder]

asyura, on 08 April 2011 - 01:19 AM, said:

protolder, on 07 April 2011 - 03:51 AM, said:

hola, your protein is insoluble if you don´t add urea, I think. why don´t you analize flowthrougt .if  the protein doesn´t bound could be because 6His are hidden, why dont you add some reducer, Nickel columns admit untill 10mM mercapto ethanol; and I think  2mM could improve your yield. Buena suerte

hye, i did analyze my flowthrough.. (after washing)..but my protein was not there..means it is stuck at the column but doesnt want to elute out once i put the elution buffer..it seems like my protein is too stubborn..bind strong to the column..i will try to put mercaptoethanol..but, i am still wonder what is the purpose of reducer. i read before about it, but i cant understand much..
thanks anyway

hola, when you says filterirg you refers to any of 0.8,or 0.45,or 0.22microns because in other filters you don´t eliminate aggregates wich are seed at the top of the colum. reducer helps to broke s-s- intermoleculars wich lead to the aggreates formation. Buen dia

[BioMiha]

I would opt for a different approach. I think your protein aggregated on the filter and did not even make it to the column. I am not sure what is the final goal of your purification but I would suppose that you would like your protein folded and active. In this case if you add urea and 2-ME you will completely denature your protein and render it useless until you perform the refolding (which is in my opinion usually very ineffective). Why don't you first try to purify your protein without filtering it. Just perform a centrifugation step where you eliminate particles and non-soluble protein and apply the clear supernatant to the His trap column. If the protein is there, you will be able to see it.

[asyura]

protolder, on 08 April 2011 - 01:56 AM, said:

asyura, on 08 April 2011 - 01:19 AM, said:

protolder, on 07 April 2011 - 03:51 AM, said:

hola, your protein is insoluble if you don´t add urea, I think. why don´t you analize flowthrougt .if  the protein doesn´t bound could be because 6His are hidden, why dont you add some reducer, Nickel columns admit untill 10mM mercapto ethanol; and I think  2mM could improve your yield. Buena suerte

hye, i did analyze my flowthrough.. (after washing)..but my protein was not there..means it is stuck at the column but doesnt want to elute out once i put the elution buffer..it seems like my protein is too stubborn..bind strong to the column..i will try to put mercaptoethanol..but, i am still wonder what is the purpose of reducer. i read before about it, but i cant understand much..
thanks anyway

hola, when you says filterirg you refers to any of 0.8,or 0.45,or 0.22microns because in other filters you don´t eliminate aggregates wich are seed at the top of the colum. reducer helps to broke s-s- intermoleculars wich lead to the aggreates formation. Buen dia

hye, i use 0.40micron syringe filter from sartorius.i dont think ke protein was aggredates on the membrane since i can get other protein in unbound fraction.if the protein stuck in the membrane, i will not get anything for the unbound right? correct me if i am wrong..thanks

[asyura]

BioMiha, on 08 April 2011 - 03:51 AM, said:

I would opt for a different approach. I think your protein aggregated on the filter and did not even make it to the column. I am not sure what is the final goal of your purification but I would suppose that you would like your protein folded and active. In this case if you add urea and 2-ME you will completely denature your protein and render it useless until you perform the refolding (which is in my opinion usually very ineffective). Why don't you first try to purify your protein without filtering it. Just perform a centrifugation step where you eliminate particles and non-soluble protein and apply the clear supernatant to the His trap column. If the protein is there, you will be able to see it.

hye, thanks for your option.i will try to run my sample without filter it first.but actually my concern is, the AKTA engineer that remind me before to filter everything before i inject to the system because he afraid of the system clot if got any particle stuck in the capillary.but i will try later.and one more,i did use the urea since my protein is inclusion bodies which it is very hard to soluble. even i put R2 also the pellet is difficult to dissolve.the final way is to put high concentration of urea.i will immunize the mice using the purify protein which i think it is not really important to keep it folded and active right?thanks anyway

[protolder]

[hola, you can have aggregates of your protein alone or mixed with others, but you can have other soluble proteins withouth aggregate wich will elute in the flowthrough. You can keep a little sample before and after filtration for analyze by PAGE, and could mesure protein in both samples to see if it has been any part lost. Buena suerte