If we store the protein and we have to measure the weight of tissue and after that we stored again.
May be ones is too much! It depends on the protein, therefore one has to check the behaviour of its own protein.
I agree with Giancarlos: some proteins might be already degraded or altered after the first thawing. Most proteins survive one freeze-thaw cycle, but since during the freezing and thawing quite some cell structures are destroyed, degradation will accerelate. So I would recommend not to refreeze a tissue sample, but rather to process it (e.g. homogenize in a buffer containing protease inhibitors).
Agree with both. If your protein is stable, you can rely on one freeze-thaw cycle. If its unstable, it wont even survive once. Its best not to put all your eggs in one basket!!
For activity studies of TGF beta 1 like using ELISA, cannot thaw tissue for more than one time.
It is more stable in whole cell lysate than in tissue. In tissue it may undergo proteolytic cleavage after thawing.
I would recommend you to isolate whole protein lysate from tissue and make aliquotes for activity studies. If you would like use acid activated TGF beta 1 for an assay, keep it on ice and use it on the same day.
Hello RIKI,
I agree with all the above comments. The relation between the thawing frequency and the protein stability varies from one protein to another. In addition you would like to keep the following things in mind:
1) Thawing should happen in 4 C followed by room temperature for a gradual temp shift.
2) You should be careful with the pipetting, dont vortex and make sure that the you don't get foaming when dealing with the proteins.
3) The buffer composition, the protein composition ( the presence of metal ions in the proteins) can also tell you that what extra you should add in the buffer to keep your protein stable.
4) The tissue from which you are extracting your protein.
Hope this is of some use to you. All the best!
CHEERS!!
Jitendra
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue