DTT in protein sample for western blot

[Sophs012]

HI,

I have extracted some neutrophils, and generally make up lysis buffer with SDS buffer, DTT and protease inhibitor. Yesterday I ran out before I could add the buffer to my last sample, and thus made up some more.

There was some DTT, that was quite 'powdery' on the side (its an aliqout out the freezer) that I used in the last sample and I was wondering how much this would effect the denaturing of my protein (I'm probing for MCL-1)?

Thanks

Sophie

[BioWizard]

What do you mean powdery? Did you thaw it out and made sure it's completely homogenous and clear before you added it to the loading buffer?

[BioWizard]

By the way, the SDS is the denaturant. DTT is a reducing agent which breaks the disulfide bridges to 1) aid opening of the peptide chain and 2) prevent cross-linking. If you added enough and it was completely dissolved into the sample, you should be OK. You'll know right away when you develop your blot.

[Sophs012]

hi,

it wasn't completely homogeneous and seemed like the actual DTT (I assume is a powder that gets dissolved to form the solution) had saturated out.

Oh, of course, thanks for the clarification. I'm going to test the sample on a gel and ponceau stain the membrane, but for argument sake, could I add a tiny bit more DTT if the protein doesn't run down?

[BioWizard]

Not having a reducing agent isn't going to prevent your protein from running down. It can, however, cause some disulfide cross-linking and you end up with a laddering effect instead of a nice sharp band.

Just add a tiny bit of beta-mercaptoethanol to your sample before you load it on the gel (1% will be plenty).

[Sophs012]

just to be awkward, as I'm not sure if we have much beta-mercaptoenthanol we have (i know that has the same effect as the DTT) can I use 1% DTT - I know we have some aliquots that are perfectly fine.

[BioWizard]

OK, if you have aliquots that look fine you can just add some of that (figure out what final % you need and add enough to make that). You can warm up your frozen aliquots a little to help them redissolve if they precipitated.

You can spin your samples at 13000g for 1 min (tabletop mini-centrifuge) before loading on the gel, to pellet down any undissolved stuff..

[Sophs012]

Thanks for your help. Generally I use about 10% DTT, but assuming that there may be a bit in the sample already, should I reduce this?

[BioWizard]

What's your stock concentration of DTT, and what is your sample volume?

You don't want to dilute your sample up too much.

[Sophs012]

the DTT is 10mM and the volume of my sample is 35microlitres