Recently, I met a tricky system.
The proteins made from TNT.T7 reticulocyte lysate are sensitive to freezing and thawing steps.
I was assuming the denatured protein might be more stable than regular stuff.
But, even though I tested different ways to store them, the 2nd run for SDS-PAGE is completely different to 1st run.
I have a protease inhibitor inside. I tried to snap freeze my samples. I re-boiled my samples before loading.
Has anyone experienced something similar?
Hi,
I always had a 'feeling' that the proteins are sensitive as the outcome of the assays ( I did degradation) sometimes changed with multiple freeze thawing I dont think there is too much that can be done but I can tell you two tips that helped me.
1. I always made fresh samples for the important or new experiments, you can make them in small volumes (I did 12.5 micoL) , The whole process takes not more than 2 hours (once you know how your plasmid is translated) and with the gel half a day, so it is not that bad...
2. You can also dilute the Retic at least x2 with buffer or water and you will get the same translation efficiency so you can save on that quite a lot.
Hope it helps
Peter
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