Questions about protein gel loading and immunoprecipitation - (Dec/05/2006 )

hello everybody
i am sorry i have 2 questions :
the first is about loading the gel , i read before here that if i amnot going to use all my wells i have to add the same amount of running buffer to the empty wells and another opinion is to add lameli buffer so is the idea just to fill the empty wells in order to get nice bands at the end?

the second one about IP
In some protocols , people just use lysis buffer for washing and some use pBs so whats the difference and if i used protease inhibitors in the lysis buffer should i use it as a washing buffer?

thankxxxx

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1) I prefer loading 1X sample buffer over running buffer to fill the empty wells, roughly same amount as to your other samples.

2) The difference is minor. You can include protease inhibitors for the safe side.

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1) usually I don't load the empty wells, unless I know that my samples have a high amount of salt or detergent, then I load with my sample buffer and laemmli (it's rare, when I have 20 µL of cell lysis in 0.1% triton X-100 150 mM of salts, I don't load empty wells)
It's too avoid that the bands that contain salts become larger, and bands that do not contain salts become thinner.

2) I prefer to use lysis buffer, because there are detergents that helps to get rid of non specific interactions, especially if I detect with streptavidin-HRP captured biotinylated proteins. There I can see that I precipitate plenty of proteins if I don't use the appropriate wash buffer. Sometime I even increase the concentration of NaCl up to 500 mM.
I don't use protease inhibitor in wash buffer. it's a 4°C and quick step right before loading. Not so much risk to have degradation, and most of enzymes should be eliminated during the first wash.

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thankx guys
missele
my supervisior said that in IP dont use high salt concentration in lysis/wash buffer so i am using NP40 150mM NaCl for it and for my lysis/wash buffer for GST pulldown assay use NP40 NaCl 250mM
thankx

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Q1: loading of protein-empty wells with 1x sample buffer is more a control to show that bands in Wb are not from sample buffer rather than for better performance

Q2: washing with PBS instead of lysis buffer is for some labs a question of economy as protease or phosphatase inhibitors are expensive; for IP 150 mM ionic strength are ok, for pull-down I would optimize as protein-protein affinities vary more than in IP

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