Dear santosh,

I have also faced similar problem. I have few suggestions here:

1. First, know the optimal concentration of your protein in the current buffer, where it does not aggregate. You can do so by using dynamic light scattering (DLS) technique. in detail, while concentrating your protein after gel filtration, concentrate it  at different concentrations lets say 2,4,5, 7 8 or 9 mg/ml. And check it using DLS, which gives you the polydispersity, roughly M.W, and diameter of your protein sample in solution. Eventually you will find some concentration up to which the sample is monomer, and exhibit minimum polydispersity.

2. You can try crystallization starting with the right concentration (even if its low approx 5 mg/ml. you never know you might get crystals; good enough for diffraction.

3. You have to optimize your buffer conditions in order to keep your protein in monomer. DLS is also nice tool for buffer optimization, check detail how? You  may change your buffer content(pH, different salts, ionic strength etc.) firstly. If this does not work, you can add some additive to your buffer, like EDTA, glycerol, amino acids(arginine, glutamine etc), urea, etc.

Here is a paper showing how to prevent protein aggregation.

http://www.sciencedirect.com/science/article/pii/S0003269703000599

Good luck.

Thank you Shiv....

Hi Santhosh

I agree with the recommendations posted. However, it must be worthwhile to check for protein characteristics. I experienced similar things with RNA-DNA binding proteins, which typically like more salt. I will increase the amount of salt in your gel filtration buffer to 250-500 mM.

Best. Paco

I will go through it Francis...

Hi Santhosh,

I totally agree with the above posts.

There is a technique called thermal shift assay or differential scanning fluorimetry which gives you the melting point of the protein that in turn reflects how stable your protein in a particular condition. If you have access to that then you can try different salts, buffer conditions for the stability of the protein. Sometimes adding glycine or arginine can stabilize a protein.

Did you check the pI of the protein? You should be at least be one unit away from the pI. But may be you have already checked it.

regards,

Deep

Thank you Deep...

My protein have pI of 7.04 and iam using a buffer of  pH 8.0

Adjusting the salt concentration will help

First - My advice is definitely push your pH up. At 8.0 you are still fairly close to the pI. Having a higher pH = protein is more charged = more soluble. Especially If your protein pI is theoretical - you don't know how accurate that is in reality, and it may be closer to 8.0 than you think. If using Tris (a good idea for crystallography) then you would get away with pH 8.5 no problem.

Second - I notice you have no reducing agent in the buffer. This is fine if the protein is supposed to have disulphide bonds or has no cysteines, but if there are surface cysteines, perhaps they could be causing disulphide mediated aggregation??

Third - Why do you have a particular concentration in mind for crystallisation? It may be that you are simply approaching your protein's solubility limit - and 9.2mg/ml is a perfectly reasonable concentration for crystallography. Was the protein becoming difficult to concentrate any further? Have you put down a couple of xtal trays to get a feel of whether the protein is precipitating/phase separating? If the protein is beginning to aggregate - this might indicate that this is a good concentration to trial!

Thank a lot for ur valuable suggestions,Matthew..

1.I will change the pH accordingly.

2.I will include the reducing agent in my final gel filtration buffer.

3.After 9.2 mg my protein is stable only on ice but if i shift it to room temperature it was started precipitation and we don't have xtal trays for screening.

The temperature dependence suggests that it is indeed a pH issue.

Tris buffer is susceptible to changes in temperature, and responds such that the pH is higher when it is colder - probably this is why your protein was happier on ice.

https://www.neb.com/tools-and-resources/usage-guidelines/ph-vs-temperature-for-tris-buffer

Therefore I think changing your pH is the most important thing! I'd only add reducing agent if your protein is intracellular and you know it isn't supposed to have disulphides.

I have changed to pH 8.4 buffer but it was not effected the precipitation.

On the other side i will go through the second condition (Reducing agent) to screen it.

it's protein dependant... You should check in  which buffer your protein is stable (by thermal shift assay for instance) and also try additives (glycerol, DTT, EDTA). Sometimes working at low temperature can be very usefull....good luck!

First of all you cannot work in darkness. have the sequence? analyze it. Calculates isoelectric point, net charge, ratio of hydrophobic and charged, presence of cys, in short, try to understand what have on your hands. You can obtain a lot of information that will be useful to avoid errors or the trial and error which is time as well as protein wasting. From the sequence try to determine whether the protein is globular or intrinsically disordered, because structurally the difference is large. 9.5 mg/mL is a concentration which can easily lead to irreversible aggregation.  You probably have less trouble diluting.

Good luck

Calculating the isoelectric point (pI) and keeping your buffer more than 1 pH unit away from the pI is important and should almost always be done. But appropriate buffer pH is not always enough. Additives that stabilize your protein can be used, but must be chosen carefully and tested. Glycerol is sometimes used, but I would avoid this as it typically interferes with crystallization efforts and significantly slows centrifugal concentration.

Supplementing your buffer with amino acids such as arginine, lysine, histidine or glutamine have been shown to be effective in stabilizing proteins (see Falconer, R. J., Chan, C., Hughes, K., & Munro, T. P. (2011). Stabilization of a monoclonal antibody during purification and formulation by addition of basic amino acid excipients. Journal of Chemical Technology and Biotechnology, 86(7), 942–948. doi:10.1002/jctb.2657 ).

Adding these amino acids has helped to stabilize my proteins in the past. 

Also, a mixture of arginine and glutamate has been used effectively in NMR studies ( see: Hautbergue, G. M., & Golovanov, A. P. (2008). Increasing the sensitivity of cryoprobe protein NMR experiments by using the sole low-conductivity arginine glutamate salt. Journal of Magnetic Resonance, 191(2), 335–339. doi:10.1016/j.jmr.2007.12.017 )

For crystallography, adding amino acids up to ~50 mM may be acceptable, as at these concentrations they will serve as "additives" to your crystallization screen. Please note that this will likely significantly alter results and any published crystallization conditions are likely no longer relevant. However, published conditions are moot if your protein crashes out prior to setting up crystallization trials. 

Thank you,Carolie,Giovanni and Richard for your valuable suggestions...

My first choice, with out more information, will be increase the NaCl concentration (Salting-in effect) and avoid at this level additives than could interfere later with the crystallization (another variable to check). Also check your pI, as was mention before. You can already try your first crystallization assays, or try at 5.0 mg/mL for instance.

Hi Santosh,

Happy to see your query here and how do u do?

I agree with all of the above valuable inputs but still I am not clear with query. If I am not wrong in understanding that after gel filtration again you are concentrating  monomer fraction protein to 9.2  mg/ml and reloading onto gel filtration to check homogeneity.

Why you need a second round of gel filtration, for crystallization one round of gel filtration will sufficient to serve as a final polishing step(if your end up with >95% purity or else you have to look for some other chromatographic method) 

If your taking monomeric fraction a  SDS gel is first and good validating tool  to check purity of protein no need to worry about homogeneity. In the process of concentrating protein to lower volume you are pushing protein to a less soluble state so obviously it will aggregate.(as a simple logic individual monomeric fractions after GF are soluble that means at  diluted state your protein is happy but if reaches beyond its concentration limit to exists as soluble it tends to insoluble forms...viz aggregation, precipitation).

From your query here I have some more inputs

>I am not agreeing pH is a parameter in this regard because till gel filtration it's soluble means that is a happy buffer condition for your protein no doubt but after that concentration is the observable parameter to optimize your protein solubility.

>Are you spinning your sample at ~10k for 10min before injecting onto GF column? if not obviously you will find aggregate in your chromatogram (that is insoluble content in total mixture).

Hope I have not perplexed to you, 

Good luck

Hello eswar glad to see your reply,

1.I have done GPC before crystallization just as an optional to know whether my protein is forming some oligomers or not after 9.3 mg.

2.Before injecting sample it was centrifuged and filtered through the 0.2um filter.

I've usually just diluted it till it doesn't crash out, or used whatever was left after the crashing out, or in very rare cases added detergent if really needed. You want it to crash out very slowly as a crystal anyways, so unless the protein is hard to make I would go ahead and start crystal screens and adjust dilution based on your screen scores instead of worrying about long term storage.

Expanding slightly on the first suggestions. You can use dynamic light scattering (DLS) to find a more suitable buffer by checking for lower polydispersity. This could be done at relatively low concentration to screen pH, additives, temperature, and then also look at concentration effects. Statistically crystallization success is more likely when starting from a homogeneous, low polydispersity protein than from an aggregated or even oligomer / monomer equilibrium starting point. Good luck!

http://www.materials-talks.com/blog/2014/10/23/polydispersity-what-does-it-mean-for-dls-and-chromatography/

http://www.malvern.com/en/support/resource-center/application-notes/AN101104CharacterizationRiceDwarfVirus.aspx