Is adding reducing agent necessary before protein boil? - For Western blot (Jan/31/2005 )

Hi ,

Does anyone know if it is a must to add reducing agent to the protein sample buffer before boil?

I had my lysis buffer added with 2mM of DTT. Do I still need to add anymore reducing agent to the protein lysate and bring it to boil before losding on to a gel?

Thanks a million,
Joey

Hi
tells us more about your experiments in order to know were did you get your proteins (yeast production, extraction from bacerias, cells or yeast...)

Here is the protocol I routinely use for mammalian cells protein extraction :

use 300µl of lysis buffer for about 5 millions of cells
incubate cells + lysis buffer 10' on ice
Centrifuge 14000 rpm 30' 4°C
discard the pellet
the extract is ready for quantification and more analysis

sample buffer :

NaCl 150mM (3ml NaCl 1M)
EDTA pH8 2mM (160µl EDTA 250mM)
NP40 1% (2ml NP40 10%)
Tris HCl 50 mm pH 7.5
ddH2O qsp 20ml

just before use add for 1ml of buffer :
-5µl DTT 1M (to get 5mM)
-1µl PMSF 100mM

I don't know if more reducing agent is necessary. All can i say is that these conditions are very good for me (on total cellular extract analysed in SDSPAGE and for western blotting).
For your experiments, why don't you try these conditions and check which is the more suitable for you?

You can also make several concentrations of denaturing agent (but i don't think that more than 10mM is necessary).

hope that will help.
keep us informed of your experiments

Hi

I'm using the lysis buffer containing the components below for extracting proteins from mammalian cell lines:

Tris-HCl pH8 10mM
TritonX-100 0.10%
MgSO4 10mM
CaCl2 2mM

Micrococcal nuclease 75U/ml (freshly added)
DTT 2mM (freshly added)

50 to 100ul is added to each pellet and freeze thaw vortex for 3x.

5x Sample Buffer

Glycerol 62.5%
Tris pH6.8 160mM
SDS 5%
Bromophenol Blue 0.1%

beta-mercaptoethanol 5% (Freshly added) -Which I don't know whether to add anot.

Boil for 5mins, spin down at 10x1000rpm for 5mins before loading.

The reason for the question of whether to add reducing agent in the sample buffer is because I once did a westernblot for a membrane protein using the method mentioned above. I can only get my band of interest when I exclude beta-mercap in the sample buffer.

Does your method feasible for extracting membrane proteins? 'Coz mine is a protocol passed down from my boss.

Your help is greatly appreciated! Thanks alot~
Cheers

Hi. DTT is used in most lysis buffers like yours at low concentrations (1-10 mM) to stabilize proteins with free sulfhydryl groups. When you use it as a reducing agent in the SDS-PAGE sample buffer instead of 2-mercaptoetOH you need to add a higher concentration, usually around 50 mM. I lysate my cells in a buffer containing 1 mM DTT and then add a sample buffer with mercapto or more DTT for running them in PAGE. However, it is not a must to add a reducing agent in the sample buffer, only if you want to dissassemble the proteins into their subunits. Other thing is that membrane proteins, specially high MW ones are sometimes tricky and is better not to boil them, but heat them at 37ºC, in the presence of DTT or mercapto if you want to dissociate them. Hope this helps!