Difference between revisions of "Cell Transformation Group:Protocols/Protein"

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Line 19: Line 19:
 
{|border="1" cellpadding="5" cellspacing="0"  
 
{|border="1" cellpadding="5" cellspacing="0"  
 
|-
 
|-
!colspan="2" align="left" |Cell Lysis Buffer
+
!colspan="2" align="Centre" |Cell Lysis Buffer
 
|-
 
|-
!Concentration!!Reagent
+
!Concentration!!style="text-align:left"|Reagent
 
|-
 
|-
 
|10mM||Tris HCl pH8
 
|10mM||Tris HCl pH8
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Nb – Complete is stored at 25x concentration<BR>
 
Nb – Complete is stored at 25x concentration<BR>
 
Add:<BR>
 
Add:<BR>
:40μl 25x to: 1ml volume <BR>
+
:40μl 25x to 1ml volume <BR>
:2μl 25x to: 50μl volume<BR>
+
:2μl 25x to 50μl volume<BR>

Revision as of 15:59, 15 January 2009

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Harvesting Cell Lysates for Western Blotting (NEW METHOD)

(Takes about 1-1.5 hours)

1. Harvest cells and supernatant into a Falcon tube by trypsinisation
2. Use aliquot of supernatant to collect remaining cells in flask or 6-well plate. Transfer to appropriate tube
3. Spin for 5-10mins @ 250g (turn on 4°C centrifuge)
4. Discard supernatant into Virkon
5. Resuspend cells in 1ml of PBS and transfer to a tabbed, eppendorf tube
6. Remove a 20µl aliquot for counting
7. Spin for 10 mins @ 3000rpm/4°C (get ice)
8. Discard supernatant and resuspend in appropriate volume lysis buffer (+1xComplete) to resuspend at 2 x 104 live cells/µl or other useful concentration depending on expt
9. (can do this now or later) Lyse cells for 30mins on ice, mix, spin cell debris down, transfer supernatant to labelled eppendorf
10. Store at –20°C


Cell Lysis Buffer
Concentration Reagent
10mM Tris HCl pH8
150mM NaCl
1mM EDTA
1% NP40
0.1% SDS
1x Complete protease inhibitor

Add Complete just prior to use
Nb – Complete is stored at 25x concentration
Add:

40μl 25x to 1ml volume
2μl 25x to 50μl volume