I am trying to look at protein in a non-cellular structure using SDS-PAGE. I will be collecting plenty of those structures from the model organism on different days. Is it wise if I collect structures of one day, keep them in sample buffer, at -80 degree until all the sample have been collected for SDS-PAGE, ultimately.
Yes, if you are thinking just to do an SDS PAGE with your samples, you can flash freeze them and then put them at -80°c. If you want to do other applications (activity assays etc.), you 'll have to be a bit more careful.
How stable are these setae?
You may have to optimize your buffer for freezing.
For instance, don't use tris, as it changes pH strongly with temperature. You may have to add sugars or glycerol.
I would first try a small experiment only with the buffer of your choice and analyze split samples kept for hours, days, many days before analyzing them. Do you get the same results after different hold times of the same sample in the freezer?
Achim Recktenwald , Previously - when I couldn't see any protein runs in the sample - I had isolated ample numbers of setae, transiently stored in PBS in an 1.5mL eppendorf, replaced with sample buffer, later, and stored in -80 due to certain reason. I performed SDS-PAGE about a week later to see no protein runs. Where did it go wrong - keeping in sample buffer for a week at -80 or using tris that changed pH at -80 (as per your answer). I would like to know if by adding glycerol, I can keep seate in the sample buffer for couple of days(collecting all the sample in a day isn't possible).
There may be lots of protease activity on the setae. Try adding protease inhibitor cocktails. They are commercially available.
The other possibility: the amount of proteins may be very low. How did you stain? Just Coomassie or did you try silver staining?
I would perhaps also try vortexing the setae kept in sample buffer after thawing them. I would do a strong vortexing.
Are the setae made mainly of protein or some other material, like chitin?
- Badges
- Science method