Making W.Blot Samples

Hello all,

  A co-worker of mine and I are debating the most efficient way of making our protein samples for Western Blots.  She is convinced that heating at 50-70C for 10 minutes w/SDS is sufficient.  I am convinced that samples need to be heated to at least 95C for 5 minutes w/SDS before proper denaturation can occur.  The only reason I (personally) would use a slightly lower temperature is to avoid aggregation of multi-pass membrane proteins.  Does anyone have any insight on this so we can settle our dispute?  Thank you! 

Both way should be good enough to denature proteins. And many believe that 70C is better.

beside the reason you listed, another advantage for 70 is that your eppendorf won't pop open during the heating.

I myself prefer 70C, because of the reason above, and also because I can keep a heat block (water both) at 70 all the time.

I think it would be better if one uses a temperature aroung 70C so as to take care of most of the samples. The time should be atleast for 10 mins and max 20 mins not more than that otherwise many proteins will be denatured.

Hi kgambles,

Remember that your protein solution contains SDS and likely other denaturants, so there really is no need to heat the samples as high as 95oC. The combination of 70oC and denaturants for 10 minutes should be enough to denature a reasonably concentrated protein solution. 

'Most' proteins are denatured at 41C, so 70 or 95 will have essentially the same effect on the majority of proteins assuming you are using DTT to denature S-S bonds. This is because the hydrogen bonds that make up the majority of structural support for proteins are relatively weak and are broken with moderate stresses like added temperature.

Of course, if you are working with a Thermophilic bacteria all bets are off! Thats why of course Taq DNA Polymerase is able to do its thing. Sorry I had to throw that in, because I was questioning myself on that one.

Rus

My experinece is explicitly that multipass membrane proteins really DO NOT like to be boiled or heated at all during denaturation for western blots and you get massive aggregation in the wells if you do heat them.

70 C is better and more time of heat incubation or temp more than that leads to aggregation like phnomenon to happen