Degradation is probably caused by contamination with proteases. You should use protease inhibitor cocktail during extraction and purification, and get the protein as pure as possible. Work at 4oC and keep all protein fractions on ice during purification.

Precipitation may be prevented by adjusting the pH and/or salt concentration, by reducing the protein concentration, or by adding a mild detergent. This assumes that the protein is properly folded.

If the protein is partially or completely denatured, this would probably result in precipitation. You might be able to stabilize the protein through extraction and purification by adding a ligand, such as a substrate.

Degradation is probably caused by contamination with proteases. You should use protease inhibitor cocktail during extraction and purification, and get the protein as pure as possible. Work at 4oC and keep all protein fractions on ice during purification.

Precipitation may be prevented by adjusting the pH and/or salt concentration, by reducing the protein concentration, or by adding a mild detergent. This assumes that the protein is properly folded.

If the protein is partially or completely denatured, this would probably result in precipitation. You might be able to stabilize the protein through extraction and purification by adding a ligand, such as a substrate.

hi,

degradation usually occurs during the purification step. to avoid this, use EDTA (1-5mM) in the lysis buffer and, if possible, protease inhibitor cocktail. precipitation can be caused by many different conditions, e.g. warm-cold steps or high protein concentrations. may be using some reagents such as PEG can help you keeping soluble your protein sample. hoping this will be useful! good job!

I was once told, that proteins are most stable in high concentration, which makes sense since cells are full of stuff and they are used to be crowded. That's why you should use additives such as PEG, glycerol, NaCl etc.

Maybe something else may be usefull for your protein like reducing agents, metals etc.

I agree with Adam, Sigma offers a suite of different protease inhibitor cocktails - http://www.sigmaaldrich.com/life-science/molecular-biology/molecular-biology-products.html?TablePage=14562002, relatively cheap for larger volumes especially during cell lysis.

Precipitation and degradation both depend highly on properties of the protein. In case protein surface is hydrophobic, use PEG (which also is widely used to aid in crystallizing proteins) or glycerol and container with low protein binding surface. If protein is hydrophilic, it usually does not precipitate easily, but it helps using salt in solution (can also use respective salts which help in crystallization).

Proteins are more stable at higher concentrations, but this also may lead to precipitation more easily. Concentrate the sample just before crystallization, storaging would increase the odds of both precipitation and contamination. Concentration by centrifugation is relatively risky for easily precipitating proteins, so lyophilisation is a safer way. 

In case your protein is enzyme, an affine inhibitor or ligand is a good way to promote stability and also, crystallization.

hi Manish,

Preparation of a concentrated and stable protein

sample is a prosaic but often difficult task, as proteins frequently

aggregate or precipitate at higher concentrations, and are

sometimes subject to spontaneous proteolytic degradation.

In order to avoid degradation and have high stability for protein, you can have a look on the upload documents.

Good luck

Hi,

Degradation is a common issue if your sample isn't free of proteases, but if you want to crystallize it you should consider that your purification hasn't been effective enough.

Protease inhibitors in first steps of purification usually do the job quite well (Sigma or Roche cocktails are good, but avoid those with EDTA if your protein needs divalent cations).

For your precipitation problem, it is a different matter, which needs more informations for us to advise you with some good tips (notably pI of your protein and pH of your purification buffers, need for conterions or cofactors, salt concentration of your buffers, and so many more...). Give us some more about when it does precipitate if you want us to give you good advises !

Hope this will help !

YV

 Use inhibitors and keep everything on ice, but most importantly do the purification as quickly as possible - never let a protein sit around when it doesn't have to.

Also - PMSF is only stable in water about an hour.  If that is your only inhibitor, that's your problem.

Hey Manish,

To add on to whatever everyone's been saying, it'd be good to get more information on the protein, e.g., PI, cofactors (does it need zinc in the case of zinc fingers or molybdenum in the case of proteins with MoCo), etc. 

I'd like to add that the strain that you use matters as well. Personally, I use either Rosetta or BL21 strains, i.e. opt for low protease strains.  

Work fast! Purification should be done ASAP, so as to isolate the protein of interest and then freeze it asap. That said, if the protein isn't stable to begin with it might degrade during crystallization trials anyways.  

Degradation could imply that you might need to change to a different construct as well, i.e. lengthen it or shorten it. Or add glycerol and increase salt concentration. That could work. What's your current buffer?

Hi Manish.

As many people suggested, use the commercial protease inhibitors cocktails (Roche has a broad selection of them). Someone suggested to add EDTA to the buffers, that's a good idea. Just keep in mind that sometimes "high" concentration of EDTA are not compatible with some columns (Nickel columns for example).

I normally add 1mM of PMSF and 1mM of Benzadiazemide, protease inhibitors that are not present in the cocktails sold by Roche.

Good Luck!

Degrade can mean different things.  

If you suffer from proteolysis, the previous answers are spot on.  You should also improve your purification.

If your protein is unfolding over time and then precipitating, you need to stabilize its fold.  Do try osmolytes.  Up to 50% glycerol is a great standard but high glycerol must be removed prior to crystallization trials.  Do not freeze thaw your protein.

Do you use any kind of protease inhibitors or you do not? Is this a hypothetical question or you are actually experiencing this despite all your precautions? So, it all depends on your answer to these questions. If you are not using an inhibitor cocktail of some sort of course you will see degradation; so start using. Good luck!

After which purification step does your protein precipitate? Which buffers are you using for purification? I suggest you do a buffer screen for your protein to check which buffer conditions (ionic strength, salt concentration, pH) are best for your protein.