Hi guys, I have recently optimized my protein expression from BL21 cells and I have a query on the types of storage methods that can be used. I have seen the use of 0.02% sodium azide and 50% glycerol but am not particularly sure if there is a general rule on the types of components that have to be added. My protein is currently in Tris-Hcl buffer (pH8.5). Desalting has been performed to remove imidazole and NaCl. I will need this protein for downstream assays so its activity is important. Please advise on the composition of storage conditions and buffers that you guys have tried. Also, is salt (such as NaCl) required in the storage of proteins? Thanks!
The storage protocol depends a lot on the particular protein you have to store. First of all you should know if the protein loose its activity upon freezing, in this case better to add glycerol (50% is enough) before freezing them,. otherwise you may keep it at 4°C in the presence of sodium azide and ammonium sulphate at the higher concentration not affecting the solubility or activity of the protein. .I hope this will be useful for you
Hi there,
I agree with Paul for long-term storage flash freezing is the best. I would add that the best is also to check protein homogeneity after thawing (gel filtration ) for comparison with the sample quality before freezing step.
Flash freezing in liquid nitrogen in presence of a cryoprotectant (e.g. 20-30% glycerol), moderate concentration of salt (20-50mM NaCl, and bacteriostatic agent (such as sodium azide) is the first step. Quick thawing when you take it out of storage helps. However, proteins vary a lot in stability and testing the activity, aggregation, and oligomeric state before and after any attempt to store is needed to confirm the effect of storage. Some proteins, like immunoglobulins stay fine for extended period even at 4 ºC, while others require lyophilization.
when you know the optimal conditions for its activity, you should avoid them for your storage. so, i use a pH out of the optimum and higher salt concentrations. 10 - 50% glycerol is also useful but then you dilute your solution. so, it depends on your assays. also, avoid azid when you want to use it in cell culture or in vivo studies. i hope this is useful for you.
widely used methods: 1) freezing samples in liquid nitrogen: adjust pH and salt concentration, it depends on your protein. add 5%- 20% glycerol to your sample before you freeze them in liequid nitrogen and then store them at -80°C. 2) adjust pH and salt concentration and add to 50% glycerol, store samples at -20°C directly (don't freeze them in liquid N2). 3) lyophylization. and store at 4°C or -20°C.
PS: TRIS, HEPES and Phosphate buffer have difference to the protein-stability in storage. KAc or NaCl can be used for storage, it depends on proteins.
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