We don't know whether the agent will interfere with a translation assay we want to do. Would proteins usually get dentaured if they are snap frozen in a buffer without stabilisation?
1. Relatively small molecules and short peptides generally tolerate freezing well. Though even here, I would tend to aliquot the preparation so that it is thawed a minimum number of times. Larger proteins (>10 kD) may be more sensitive, particularly to repeated thawing. It is thus best to test whether the protein will remain functionally competent after 1 freeze-thaw cycle. Repeated freezing and thawing is asking for trouble.
2. When storing proteins in 50% glycerol, I would expect to dialyze the glycerol out of the storage solution prior to use. I would not expect to just use it with glycerol, unless it is at the very least very diluted (at most a few percent) in target buffer.
3. Some proteins will keep for a while at ~4C as suspension of ammonium sulfate precipitates. Then, prior to use, an aliquot would be microfuged to separate the AS solution, followed by solubilization of the pellet in target buffer.
it depends, if the freezing has a storage role, the buffer is sufficient, it is not necessary, except in special cases to add an agent such as glycerol.
by cons if the purpose of freezing is to make an X-ray analysis of your protein, you should use a cryo protectant such as glycerol, sucrose, or paraffin oil for a glassy phase.
glycerol and sucrose then replacing the water molecules to prevent the formation of ice, which would disrupt your analysis.
Thanks! I thought stabilisers were fairly routine but I shall try leaving it out. Forgot to add that I may be freezing in liquid nitrogen so I hope the protein will be ok.
The buffer including the protein is sufficient, you may store it for freezing and for further purification or either go for crystallization methods.
Depends on protein. Some will be perfectly ok, some will denature if there is no glycerol in buffer. Also optimal glycerol concentration will vary for different proteins. I was using 10% glycerol for wheat eIF4G as part of storage buffer. Also there should be no damage to in vitro translation reaction if your glycerol won't reach there more than 5%.
make sure you do not get PPT after storage in buffer. This happens when you thaw it
Hello Nandita,
In my experience, several proteins that I purified (either as overexpressed recombinant proteins or from tissue extracts) did precipitate when put to freezer without glycerol or sucrose. However, I have never tried freezing the protein in liquid nitrogen before putting it to the freezer. I heard that it may do the trick and prevent precipitation.
Good luck!
Leszek
As for most science questions the answer is "it depends". Some proteins tolerate freeze-thaw just fine, others denature or aggregate. It is rare for proteins to not tolerate storage at -20C in 50% glycerol. But cryoprotectants can play hob with downstream use, e.g. crystallography or kinetics or calorimetry. We always test small aliqouts for long term storage before selecting a method. Also, consider that many proteins tolerate aqueous buffered storage at 4C for years. Just include EDTA or some other compatible antimicrobial to prevent mold growth.
the kind of buffer and pH your protein stable is, is sufficient for storage purpose., it is not mandatory to add glycerol etc prior to storing as in my lab we used to store protein directly in buffer.
I do agree with the statement of Chandra Nath Roy and Vandana Sharma. Of course, it depends on the stability of enzyme in a specific buffer at a specific pH. The stabilizing agent, principally is not mandatory, but is needed to stabilize the activity of an enzyme if heat labile and is subjected to store at -20-80 degree centigrade for longer period of time. Further, on safer side, if 10% (v/v) glycerol is added to the enzymatic mixture in defined buffer, and stored at -20-80 degree centigrade, the subsequent freezing and thawing will not affect the enzymatic activity regardless of enzyme being heat stable or heat labile.
Try to store preps in high enough (3-5 mg/ml prtoein) concentration, allowing for significant dilution downstream. 10-20% glycerol prevents ice-crystal formation when stored in -20C. Soluble enzymes are stored long-term in 50% glycerol. After all, once you dilute glycerol concentration to
Stabilizers can be used to prevent aggregation of the protein in solution form. ...
Alternatively, glycol and sorbitol may also be used. But remember, stabilizers should be used only after assessment of the physico-chemical nature of enzyme protein.
Yes, glycol could be better choice than glycerol and trehalose is good idea as additional protectant. Also as someone pointed out keep stock protein in high conc (1-5 mg/ml - not too high too - that can induce ppt too).Some proteins may need EDTA/EGTA, some not, The same with DTT. To find the best storage buffer you really need to test combination of all these factors.
Further, it is better to use 10% (v/v) glycerol at safer side when enzyme is in solubilized state. As far as sucrose is concerned, it is restricted to use. In case of enzymes that involves carbohydrates as substrate(s) or producing carbohydrates, sucrose should not be employed as stabilizing agent just to check an biochemical interference.
Balancing of buffer system is very important for isolation and purification an enzyme, regardless of whether that one is membrane bound or solubilized. There should be perfect combination of all the ingredients as briefly shown by Krzysztof Treder.
All my proteins are stored in 10% glycerol (final concentration) and then flash-frozen using liquid nitrogen. Generally, stabilizing agents do not hinder your protein activity.
Before adding glycerol you also need to check, if glycerol interferes with your assay downstream. If it does, anyways you can not add glycerol to the protein.
1. Relatively small molecules and short peptides generally tolerate freezing well. Though even here, I would tend to aliquot the preparation so that it is thawed a minimum number of times. Larger proteins (>10 kD) may be more sensitive, particularly to repeated thawing. It is thus best to test whether the protein will remain functionally competent after 1 freeze-thaw cycle. Repeated freezing and thawing is asking for trouble.
2. When storing proteins in 50% glycerol, I would expect to dialyze the glycerol out of the storage solution prior to use. I would not expect to just use it with glycerol, unless it is at the very least very diluted (at most a few percent) in target buffer.
3. Some proteins will keep for a while at ~4C as suspension of ammonium sulfate precipitates. Then, prior to use, an aliquot would be microfuged to separate the AS solution, followed by solubilization of the pellet in target buffer.
In my opinion based on experience does not interfere. For further information, viscosity factor should be considered while assaying the enzyme protein in soluble form. If 10% glycerol is in storage condition, definitely there would be less than 1% v/v in the final assay mixture, likely to be quite insignificant as far as interference is considered. In addition, the glycerol concentration in control and experimental testing samples should be the same just to nullifying the impact of glycerol. Conclusively, it can be checked depending the type of enzyme protein. it is not at all to worry about. Further queries are welcome in future as well.
Check that, it could help:
Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions.
http://www.ncbi.nlm.nih.gov/pubmed/12673768
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