Koeris/Notebook/2007-1-24: Difference between revisions

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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.
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
|-
! NuPAGE reducing agent !! 1ul !! 1ul  !! 1ul !! 1ul
|-
|}


'''Loading of samples''' was a big problem, they were gooey and didn't want to go into the wells. Look up sample prep protocols for bacteria.
'''Loading of samples''' was a big problem, they were gooey and didn't want to go into the wells. Look up sample prep protocols for bacteria.


Preparation of whole cell. and soluble protein extract
 
===Quick and dirty protein expression sample prep===
#transfer volume X into 1.5 ml tube, X = 0.25 ml / OD
#spin 30 sec
#resuspend pellet in 30 µl H2O
#add 3 µl 1 M DTT and 11 µl 4 x SDS Loading Buffer (i.e. 10% SDS)
#5 min at 50ºC
#2 min at 100ºC
#spin 5 min
 
===Preparation of whole cell extract===
*thaw 96 bacterial pellets room temperature
*thaw 96 bacterial pellets room temperature
*resuspend in 100 µl Lysis buffer (50 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 1 mM EDTA or 0.1 mM EDTA, if NiNTA purification follows)
*resuspend in 100 µl Lysis buffer (50 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 1 mM EDTA or 0.1 mM EDTA, if NiNTA purification follows)
*add 25 µl of
*add 25 µl of


{| border="1"
{| border="1"
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|-
|-
|}
|}
*vortex briefly, incubate on ice 30 min
*vortex briefly, incubate on ice 30 min
* add 25 µl of
* add 25 µl of


{| border="1"
{| border="1"
Line 49: Line 49:
|-
|-
|}
|}
*vortex briefly, incubate at room temperature 30 min
*vortex briefly, incubate at room temperature 30 min


Whole cellular extract
===Whole cell extract===


* put 15 µl of  
* put 15 µl of  
Line 70: Line 72:
==SDS-PAGE==
==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).
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).
===NuPage sample preparation (post-extraction)===
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
|-
! NuPAGE reducing agent !! 1ul !! 1ul  !! 1ul !! 1ul
|-
|}

Latest revision as of 22:22, 24 January 2007

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.


Loading of samples was a big problem, they were gooey and didn't want to go into the wells. Look up sample prep protocols for bacteria.


Quick and dirty protein expression sample prep

  1. transfer volume X into 1.5 ml tube, X = 0.25 ml / OD
  2. spin 30 sec
  3. resuspend pellet in 30 µl H2O
  4. add 3 µl 1 M DTT and 11 µl 4 x SDS Loading Buffer (i.e. 10% SDS)
  5. 5 min at 50ºC
  6. 2 min at 100ºC
  7. spin 5 min

Preparation of whole cell extract

  • thaw 96 bacterial pellets room temperature
  • resuspend in 100 µl Lysis buffer (50 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 1 mM EDTA or 0.1 mM EDTA, if NiNTA purification follows)
  • add 25 µl of


1x: vol. per one MP well Volume
lysozym 10 mg/ml 5ul
1% Brij58 12.5 ul
Lysis Buffer 7.5ul


  • vortex briefly, incubate on ice 30 min
  • add 25 µl of


1x: vol. per one MP well Volume
1 M MgCl2 0.3ul
Benzonase gradeII 25 U/µl Merck 0.1ul
50 mM Tris-HCl pH 8.0 24.6ul


  • vortex briefly, incubate at room temperature 30 min

Whole cell extract

  • put 15 µl of
1x: vol. per one MP well Volume
4x SDS loading buffer 7.5ul
1 M DTT 2.22ul
H2O 5.25ul
  • resuspend lysates, add 15 µl to the plate, mix by pipetting
  • 5 min 50°C, 2 min 100°C
  • load 7 µl on SDS-PAGE

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).

NuPage sample preparation (post-extraction)

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
NuPAGE reducing agent 1ul 1ul 1ul 1ul
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