Increase the conc of NaCl to 0.5M along with this include 2M urea in your buffer system

Do you heat up your elution buffer? If yes, high temperatures can expose hydrophobic residues and lead to aggregates, so stick to 37o max.

It sounds like you're eluting off a Ni column of some sort. This happened to me with one of my preps and here's what worked for me: in the fraction collection tubes (at least for the fractions you suspect will have the highest concentration of target protein) initially have them containing half the volume with buffer containing NO NACL and NO IMIDAZOLE. Then add only half of your original collection volume to those tubes. This minimizes the protein concentration in the collection tube and decreases the salt concentration. Both help to disfavor aggregation.

If your protein is robust you can also consider a mild denaturant but then you would need to have another step in the purification to remove the denaturant.

Hope that helps!

You can try to have 50 mM mixture of L-Arg and L-Glu as additives to your buffers - that sometimes helps to reduce aggregation... See "A simple method for improving protein solubility and long-term stability" in JACS (2004). 126(29), 8933-9. | PMID:15264823 | DOI:10.1021/ja049297h

Thanks a lot for your advice.

I shall start working on the suggested optimisation and shall inform the method with which I am able to get the good concentration of my protein.

Regards,

Kritika

add 1 mM DTT if the protein has cys-cys bridges

I think that following these adivces would definitvely help you:

First, check the pH during lysis of your bacteria, if there is less than at least one unit of difference with the pHi of your protein of interest then she will aggregates.

second, start to protect your protein from the beginning of the experiment by the addition of 5mM of methione and Arginine in your lysis buffer.

always work on ice, with new buffers

third, you can also try in phosphate buffer.

if you need furhter informations let me know

Hello All,

Thank you for the reply.

I tried adding 1mM DTT and 50mM L-Arg in the elution buffer ( 300mM NACL, 250mM Imidazole and and 50mM NAH2PO4.) . The aggregates were not formed with the help of additives. However, after dialysis the concentration of the protein was reduced. The concentration of protein before dialysis was much higher than after dialysis.

I was wondering what could be the reason behind it.???

Thanks in Advance

Kritika:)

More likely you have impurities of proteases which cut down your protein therefore try protease inhibitors during cell rupture.

Hi,

Thank you for your reply.

I did add protease inhibitor during cell lysis and performed the purification in the cold room.

Do you think I should start the bacterial culture with larger volume ( I used 250ml of bacterial culture for protein production) ?

Regards,

Kritika

The initial bacterial volume can be increased as much as your column binding capacity.

I am not sure that you have time to optimize your fermentation conditions, if you have it then instead of bacterial volume you can increase cell concentration by using higher concentration of protein nutrients in medium and it is TB-medium.

I assume you have induction, if you have it then time and concentration of the inducer is important factors, in general the induction time should be at OD

Which buffer do you dialyze it into? You may find that having 50 mM Arg+Glu in the dialysis buffer will also help. 50 mM L-Arg is not as efficient as the mixture of 50 mM L-Arg with L-Glu. Protein may disappear due to proteolysis (in which case having protein inhibitors would help), or because it aggregates on the dialysis membrane (in which case optimizing buffer would help).

hello all..

Thank you so much...

I am finally able to get my protein purified successfully..

Many Thanks once again.

Kind Regards,

Kritika

Kritika

tell us how, please

The purification method which worked for my protein:

The protein has a his tag which need to be purified from E.coli under native conditions ; so I was follwing and troubleshooting the protocol mentioned by Qiagen. The composition of lysis buffer was same as mentioned in the protocol only with the addition of 2x EDTA free protease inhibitor. The composition of wash and elution buffer was same as Qiagen protocol ; only with some addition in the elution buffer which was 1mM DTT ( as kindly suggested by Paola) and 50mM L Arg, ( kindly suggested by Alexander) .

The bacterial cells were induced (OD : 0.6) with 0.5mM IPTG at 20 degrees for 4 hours

Dialysis buffer used : 1x PBS and 150mM Nacl

Hope that helps.

Thank you once again

All the best

Kritika:)

Thank you Kritika now your experience is useful to us all