freezing after reducing and boiling - (May/20/2009 )

just to make sure... if I prepare my loading samples adding SDS or LDS sample buffer and B-mercaptoethanol and boil them, can I freeze them and use them after defreezing without heating again? If i want to use them next day, do Ihave to freeze them or could I just leave them in the fridge?

I've been told that once u boil ur samples in sample buffer, u can leave it at RT for a few days. Now, I've never tried that, but I assume leaving in the fridge should be fine.

Tfal on May 20 2009, 11:11 AM said:

I agree...I leave the samples at RT, nothing happens in most of the cases.

Best,
TC

medchemgirl on May 21 2009, 01:17 AM said:

If you freeze them, boil them again for 2 minutes before to load

Tfal on May 20 2009, 10:11 AM said:

freezing the protein sample after addition of dye does not give any distinct advantage. u can just store it in the fridge, anyway ur proteases (if any) would have been denatured by the dye. i have left my samples at RT for 1-2 days, nothing happens.

thanks everyone.

I will from now on reheat/boil for 2 min after defreezing.
I usually leave my denatured samples ON in fridge for use next day ( I might leave them at RT from now on) while I freeze extra volume for future blots.

Now I wonder, if I load my samples shortly afer boiling, how much do I need to let them cool down before loading them?

thanks again,
Tfal

Tfal on May 23 2009, 09:02 AM said:

hey, u dont have to wait for them to cool down, u can just go ahead! at least dats d way i do n hv no prob.

YES, cool them down, if you don't much of the water will have evaporated during the boiling process and will make loading quite difficult.

After boiling my samples i do vortex for 1 second then centrifuge at RT for 1 minutes then load it.

I prepare my sample in bulk, ie dilute out all lysates to same conc, then boil with 6 x buffer and keep in -80. Whenever i need to run my gel, they are ready to load. I am anticipating using them within 6 mo at the max. Is that ok ?

I do so coz my postdoc told me it is that the denatured samples are more stable than the lysates.