Koeris/Notebook/2007-1-24

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 1: Line 1:
__TOC__
__TOC__
 +
=Expression of [His]<sub>6</sub> constructs
 +
==Sample preparation==
 +
Frozen samples were thawed on ice and diluted to prepare a simple Coomassie-stain gel check. Is the overexpression visible? If so it might be too much for a physiologically relevant pulldown.
 +
===Sample setup===
 +
Heat samples for 2min at 85 deg C '''NOT''' at 95 deg C as we would usually do. This has to do with the composition of Invitrogen's reagents.
 +
{| border="1"
 +
! Reagent !! 1ul load !! 2ul load !! 3ul load !! 5ul load
 +
|-
 +
! Sample !! 1ul !! 2ul !! 3ul !! 5ul
 +
|-
 +
! SDS laoding buffer 2X !! 5ul !! 5ul !! 5ul !! 5ul
 +
|-
 +
! ddH<sub>2</sub>O !! 4ul !! 3ul  !! 2ul !! 0ul
 +
|-
 +
|}
 +
==SDS-PAGE==
 +
We're using the Invitrogen Nu-PAGE system with Novex bis-tris 4-12% gels' and MES running buffer to get the high resolution in the low kDa-range (as advertised).

Revision as of 16:44, 24 January 2007

Contents

=Expression of [His]6 constructs

Sample preparation

Frozen samples were thawed on ice and diluted to prepare a simple Coomassie-stain gel check. Is the overexpression visible? If so it might be too much for a physiologically relevant pulldown.

Sample setup

Heat samples for 2min at 85 deg C NOT at 95 deg C as we would usually do. This has to do with the composition of Invitrogen's reagents.

Reagent 1ul load 2ul load 3ul load 5ul load
Sample 1ul 2ul 3ul 5ul
SDS laoding buffer 2X 5ul 5ul 5ul 5ul
ddH2O 4ul 3ul 2ul 0ul

SDS-PAGE

We're using the Invitrogen Nu-PAGE system with Novex bis-tris 4-12% gels' and MES running buffer to get the high resolution in the low kDa-range (as advertised).

Personal tools