I had to boil my samples (imported radiolabelled proteins into mitochondria followed by lysing them) prior to loading on an SDS PAGE. Usually I do it for 3 mins at 96°C and it always works well. But this time, it slipped out of my mind and ended up lying at 96°C for more than an hour. The buffer was a reducing one (Laemli with DTT). Do you think it might affect my proteins or it doesn't matter, since I already want them degraded?
I will anyways load my gel soon and will see the result myself, but just curious to know if anybody had previously faced the same issue.
Boiling for an hour might concentrate the protein sample, did you notice a volume difference when you loaded? I haven't boiled it for that long.
Thanks.
Banurekha: I haven't loaded the protein samples yet, but the volume seemed to be the same. There was liquid under the lid of the eppendorf, but when I centrifuged it down, the volume amounts to the same as before (atleast visually).
no!
check once the volume before and after boiling it (after short spin) with a cheap sample. I bet you, there will be a reduction of total amount in terms of volume. dnt believe what you see..just pipette it and check how dilute/viscous/dense it is. Normall after one hour you should have only precipitate (ofcourse it is blue color, if you look with your eyes). If you still has some liquid, dont hesitate to run on gel, also you can add some more buffer to your precipitate and run them too.
Ananda Ayyappan: Yes, I still have some liquid left, maybe yes, it is not the same amount of buffer I had initially. I will anyways check the volume before I run the gel and will run it for sure to see if it works.
Well, I found out that the bands run a bit slower when compared to the standard samples on the gel (when comparing the dye front) and when I do a ponceau staining of the membrane, after a western blot, all that I see is a smear and no distinct bands as one would normally expect.
Conclusion: BAD IDEA to heat your protein samples for longer duration at 96°C.
You might have try developing your gel with Coomassie blue (even after the western transfer), there you see better than ponceaus. The appearance of thick smear/bands may be due to the concentrated proteins (just like loading the pellet fraction). You may see better bands if you dilute the sample. But ofcourse as you said, it is not a good idea to boil sample for an hour.
- Badges
- Science method