Hello,
My understanding is that protein from cell and tissue lysates degrade between each rethaw. Has anyone experienced differing protein concentrations, significant enough to affect loading volumes for western blot between rethaws? Do control bands turn out different (i.e. b-actin)?
Thanks
Yes, it does change but how much it depends on the protein. I was working with the two soluble proteins and with freeze thaw concentration changed somewhere from 20 to 30 % of the initial- I also checked it by loading on gel filtration column. It would be good idea to go with the flash freeze in liquid nitrogen with 20% glycerol in the stock solution rather than putting vial directly in -80 freezer. Not sure about b-action, but I suppose it will also change.
Hi Rami,
Best way to keep protein stocks is to aliquot into small aliquts and freeze. It is not good practice to freeze and thaw and reuse the same container of protein. The most reasonable explanations are:
1. Proteins will stick to surfaces of polypropylene tubes. During freezing and thawing, you will typically encounter losses due to protein sticking to surfaces
2. There is evaporation of liquid during freezing and storage. Concentration of proteins will vary and change due to freezing process.
So, it is best practice to aliquot and store one time usage of protein solutions that you will need to periodically go to.
Best,
Hediye.
I completely agree with Hediye. The practice of freezing and thawing the sample results in loss and degradation of proteins
As well as the answers above, do not forget to be careful when thawing. I know this is probably common knowledge, but it is useful to remember. Rapid thawing and harsh handling can damage the proteins, especially vortexing when still frozen as it shears the proteins. I work with a membrane protein and the only loss I seemed to get was through my thawing process when I was in a hurry, but the loss was always minimal. Not enough to make a discernible difference on my western blots. Always thaw gently on ice to avoid any further protein loss. Good luck.
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