2 replies to this topic

[Iwenim]

Hello to all,

I am a bit confused - to isolate all proteins from a cell line I tried until now a SDS and b-ME containing buffer and boil them for 5 min at 96 °C.
Now I wanted to change the buffer (reduction of % SDS) with my recherches I found the RIPA buffer, but no protocoll mentioned "boiling" just shaking the samples cold (15°C or less).

When  do I boil the samples and when I don´t?? THe proteins get coated on the surface of a microtiter plate...

Does anybody knows the difference and when I do what??
Thanks a lot!!!
Iwenim

[almost a doctor]

Iwenim, on 23 May 2011 - 07:00 AM, said:

Hello to all,

I am a bit confused - to isolate all proteins from a cell line I tried until now a SDS and b-ME containing buffer and boil them for 5 min at 96 °C.
Now I wanted to change the buffer (reduction of % SDS) with my recherches I found the RIPA buffer, but no protocoll mentioned "boiling" just shaking the samples cold (15°C or less).

When  do I boil the samples and when I don´t?? THe proteins get coated on the surface of a microtiter plate...

Does anybody knows the difference and when I do what??
Thanks a lot!!!
Iwenim

RIPA buffer is used for cell lysis. You do this on ice. Resuspend the cells in RIPA (or add directly to the plate) ON ice, incubate for ~5min, then spin down, and transfer supernatant to a new tube.  You then mix the sample obtained this way with loading buffer (containing SDS and b-Me) and boil.

[Iwenim]

thank you a lot!!
- I know this is for used for western.
I would like to bring the proteins on to a solid phase (multisorb-96-well plate, like it is used for ELISA).
Do you would determine the protein content after RIPA treatment but before boiling with lämmli? (cause of the disturbing SDS in this assay?) should it work?

Thanks!!!