Hi Hossein Ansariniya ,

The formulation depends on your need. In case your protein is stable and you have to use it within a span of 4-5 days or at max a week, storing the recombinant protein at 4 degrees is a better option. If you require protein for other experiments that will be undertaken successively, you should aliquot your protein into multiple vials and store it at either -20 or -80 degrees. You must ensure that your protein is stored in 40% glycerol (can use somewhere between 25-50% glycerol). You can also add some bovine serum albumin to your purified protein. It will enhance its stability. Nevertheless, storing your protein at sub-zero temperatures will definitely enhance its preservation, but also reduce its stability. During freeze-thawing, your protein may denature, undergo unfolding, and ultimately precipitate. One needs to take relevant precautions during this. Also, for long-term storage, I recommend that your cell lysate should contain protease inhibitors like PMSF, DTT, or a commercially available cocktail.

For most of the proteins I've worked with, no special formulation was necessary. The proteins were stored in a simple buffer, such as 50 mM HEPES with 100 mM NaCl at a neutral pH. Sometimes, 10% glycerol was included. The important thing is the freezing process. To prevent the exclusion of the solutes from the ice, the sample should be divided into small aliquots in polypropylene tubes, then flash frozen with dry ice (I use dry ice powder, but you can also use a dry ice-acetone or dry ice-ethanol bath) or liquid nitrogen and stored at -80oC. Liquid nitrogen storage is also good, but more cumbersome. This method preserved the activity of almost all of the enzymes I've handled even through multiple freeze-thaw cycles. Including glycerol speeds up the thawing process, in addition to possibly acting as a stabilizer.

Some proteins may be stored for a fairly long time in a buffered solution containing 50% glycerol at -20oC. The glycerol prevents the solution from freezing. If storing proteins in a -20oC freezer, make sure it is not the kind of freezer that has an automatic defrost cycle.

I would not recommend long-term storage of frozen protein solutions at -20oC because the ice sublimes from the surface over time. Solutions stored this way should be used soon.

Dear Hossein Ansariniya

there is not a common rule valid for all the proteins, dome proteins are stable when frozen with standard buffers eg, PBS or Tris, NaCl while other proteins are unfolded and addition of cryo-preservant is necessary.

In my expereince, in General the addition of 20% of glycerol is able to stabilize the protein under freezing at -20°C or -80°C. However you can try to perform some preliminary small scale stress test in the different buffers and for example compare the different DSF profiles of the protein in the different buffers.

you can find an example of it on the following link

https://www.blogger.com/video.g?token=AD6v5dzr9KqZslTx9NeEjXx1XyPti8dU15eqYoE9TT8OEDaZQcNINuDKwzCAzVXkFWZA0btipKTr81doABh-VjpprwCcRukVcLhAXE_T5BzPxYbeQQA7VMFmc4DdpjS4XbOfxkwAhIWu

avaialble on my blog, ProteoCool.

good luck

Manuele

Hi,

I quite agree with Adam B Shapiro , if you need to store for few days the 4c or in ice is sufficient(but very protein dependent!). For long term you, prepare small aliquots and flashfreeze.

What I generally do after reaching desired protein concentration after gelfiltration is to make 20-50µl sets and flashfreeze them in liquid nitrogen before storing them in -70c freezer.

When you take them for use, slowly thaw them in ice.

Some proteins dont like this either, especially if you have an enzyme it might be that you lose the activity after freezing. If you want to do crystallization, keep in might that some proteins you need to set the crystallization immediately after purification without freezing them.

Hope this helps,

Regards,

Mikko

Thanks to all dear friends.

Article Protein Stability During Freezing: Separation of Stresses an...

trehalose: a few w/w% is a cryoprotectant and a common ingredient.

surfactants: at 0.1-1% w/w passivate air-water and plastic-water interfaces that can be heterogeneous catalytic sources of aggregation for some proteins. I'm currently working with a tri-block copolymer.

sucrose, dextrose, mannose, glycine: bulking agents... I think these might be more important for lyophilization, but read up in the above article. There are other articles out there, but this will give you a start.

Another thought is to clean up your protein after thawing. I used to work with amyloid aggregation kinetics. We would remove aggregates that formed from freeze-thaw or lyo reconstitution with MWCO spin filters. I've done the same using ultrafiltration/decanting. Good luck!