Hi Ananda,

assuming that -checked by coomassie, e.g.- your protein (eluate) is clean,

or you already tried to change the imidazole washing gradient to obtain it cleaner,

more informations are needed to try to help.

which the lysis buffer? (pI of the protein?)

do you perform sonication?

which are the purification/dyalisis buffers you tried (exactly)?

which is the starting concentration of your eluates?

how much you need to concentrate them (purpose?)?

just give a look at my posts in this topic:

http://www.researchgate.net/topic/Methods/post/Protein_preservation_for_along_time_How_i_can_access_to_that_avoiding_its_coagulation_or_lossing_its_active_form

ciao

Hi Giuseppe Ossolengo,

Thanks for your questions and here my things are in detail.

No the elution profile is not clean at all, I need to do Ion exchange and size exclusion after IMAC. The protein seems to be bound with a lot chaperones and that is why I tried to remove with ATP containing wash buffer.

Ya I played with Imidazole than anyone in the world...just a joke. I put 20-30mM in my lysis buffer and washed with 50mM, and ya I found Imidazole was the reason for the immediate aggregation of my protein, while useing 200-250mM in my elution buffer, if I elute with 100% of that, then I can elute a great cloudy form of the protiein and I can sediment it in a minute :(

So I changed my way of elution with Histiding(a few time only I did) it looks hopeful since there is no visible aggregates but there is a little aggregation after a night!

Concerned to pI, it is 8 with His tag so I am using HEPES with pH7 with 500mM NaCl and 20mM Imidazole.

I prefer cell disruptor which directly lyse the cell by mechanical means.

For dialysis, I collected elutes, pooled up and used the same buffer 50mM HEPES, 500mM NaCl and no Imidazole!

I would like to concentrate to a few millilitres...1-3ml. the problem I encounter here is, they aggregate and totally block my concentrator(membrane) as well.Of course,I still have my protein left to work with.But hell lof protein is going for nothing.

I would appreciate for any ideas. :)

Thanks,

Anand

Sounds interesting I will check it out, and try, because it is binding to anion exchangers and it do contains more hydrophobic residues!

Thanks Krzysztof :)

@Krzysztof

in the second case (metal leakage), the addition of some chelating agent to the eluate during collection could help?

@Ananda

did you check the temperature of your sample during disruption? I've directly experienced a big precipitation problem (or a loss of enzyme activity) due to the sample heating in this step (maybe an "induced hydrophobicity"?). sometimes is not enough to do the disruption on ice, the best is to pre-cool the sample before...

@Krzysztof: One more query, if I understood correctly, the hydrophobicity and the surface charge of my protein is responsible for the co-elution of Ni/Co metals bound to the column, which inturn triggers the precipitaion of my eluate?

FYI, the aggregation mainy apparent in the fractions containing higher amount of eluted protein!

@Giuseppe:During diruption the machine usually kept cold with running cold (4deg)water but it will stay 10deg or more during lysis. Of course I do pre-cooling, from the step I harvest my cells. I will take care this as well next time.

So you lost the activity of your protein/precipitation problem on purification due to this issue?

Thanks,

Anand

first of all try to decrease the NaCl concentration to around 300 mM-350mM. Secondly after IMAC most of the tagged protein still looks impure so you have to do ion exchange anyway..before ion exchange dialyzed in a buffer that does not have any salt..you said the PI is 8 ! so what is the pH of your IMAC buffers??( I hope its pH is not 8 becoz it will precipitate your protein anyway, as protein has lowest solubility at its pI). SO even after decreasing the salt concntration in IMAC buffer and keeping pI thing in mind if it still precipitated then it means you dont have any option rather than keeping your protein dilute. it is one of those things where you can not do anything about. how much you are trying to concentrate?

I don't know your disruptor machine, but we ordinarily use a sonicator, and also if the sample (vial) is completely inside ice, -depending on the sonication power and time- the sample can become too hot. In the past, -for this enzyme purification- we thought that the problem was the purification/dyalisis buffer, but I recently tried the old buffers with a milder sonication step (combined with enzymatic lysis (lysozime), also), and the enzyme nicely (or better) works.