User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/05/07: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Potter-Elvehjem Cell Homogenizing</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==Summary==
==Summary==
* cAMP precipitation as described [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/09|09April2010]] & [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/21|21April2010]] with incubation of 2 hours @ 4 °C
* cAMP precipitation of D9 P28 treated with 15% CSE for RII overlay. New way of disrupting cells was used that would be more gently than the previous method.
 
==Materials & Methods==
==Materials & Methods==
===Materials===
===Materials===
*  
*Potter buffer
 
**250 mM Sucrose (8.56 g sucrose/sacharose in 100 mL)
**3 mM imidazole (300 μL 1 M imidazole in 100 mL)
*Potter lysis buffer (per mL)
**1 mL Potter buffer
**8 μL protease inhibitor mix
**1 μL NaF
**1 μL NaVO3
**12.5 μL PMSF
*8-AHA-cAMP coated agarose beads
*Cold [[PBS]]
===Method===
===Method===
{| border="1"
====Disrupting cells====
|+'''6-wells'''
#Put cells on ice
!#
#Remove medium
!<u>1</u>
#Wash twice with 6 mL cold PBS
!<u>2</u>
#Add 0.5 mL Potter lysis buffer
!<u>3</u>
#Scrape off cells and pool in 15 mL tube
|-
#Using Potter-Elvehjem Homogenizer for 10-20 strokes @ 700 RPM gently homogenize cells
!A
#Leave cells on ice @ 4 °C for 15 min.
|
#Centrifuge 15 min. @ 3000 x g and collect supernatant
|
====Incubating lysates====
|
#incubate 1.5 mL of each sample with 29.3 mg cAMP for 15-30 min. while rotating @ 4 °C
|-
#Collect 100 mL of these samples
!B
#Put 1.5 mL of each sample on 30 uL of 8-AHA-cAMP beads and incubate 2 – 4 h while rotating @ 4 °C
|
====Samples on gel====
|
#75 μL lysate was mixed with 25 μL 4x sample buffer, beads had 50 μL 4x sample buffer added and incubated @ 95 °C for 5 min.
|
#Samples were put to 8% gel together with remaining lysates on [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/05/09|Sunday]]
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|}
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|+'''24-wells'''
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==Notes==
==Notes==
==Results==
* 50 μL of beads were used instead of 30 μL
*{{todo| WHAT?}}
 
==Discussion==
*{{done}} Ow..
 
==Related==
==Related==
===Related entries===
===Related entries===

Revision as of 06:50, 7 May 2010

<html><style type="text/css"> .todo { color: red } .done { color: green} </style></html>

Potter-Elvehjem Cell Homogenizing <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Summary

  • cAMP precipitation of D9 P28 treated with 15% CSE for RII overlay. New way of disrupting cells was used that would be more gently than the previous method.

Materials & Methods

Materials

  • Potter buffer
    • 250 mM Sucrose (8.56 g sucrose/sacharose in 100 mL)
    • 3 mM imidazole (300 μL 1 M imidazole in 100 mL)
  • Potter lysis buffer (per mL)
    • 1 mL Potter buffer
    • 8 μL protease inhibitor mix
    • 1 μL NaF
    • 1 μL NaVO3
    • 12.5 μL PMSF
  • 8-AHA-cAMP coated agarose beads
  • Cold PBS

Method

Disrupting cells

  1. Put cells on ice
  2. Remove medium
  3. Wash twice with 6 mL cold PBS
  4. Add 0.5 mL Potter lysis buffer
  5. Scrape off cells and pool in 15 mL tube
  6. Using Potter-Elvehjem Homogenizer for 10-20 strokes @ 700 RPM gently homogenize cells
  7. Leave cells on ice @ 4 °C for 15 min.
  8. Centrifuge 15 min. @ 3000 x g and collect supernatant

Incubating lysates

  1. incubate 1.5 mL of each sample with 29.3 mg cAMP for 15-30 min. while rotating @ 4 °C
  2. Collect 100 mL of these samples
  3. Put 1.5 mL of each sample on 30 uL of 8-AHA-cAMP beads and incubate 2 – 4 h while rotating @ 4 °C

Samples on gel

  1. 75 μL lysate was mixed with 25 μL 4x sample buffer, beads had 50 μL 4x sample buffer added and incubated @ 95 °C for 5 min.
  2. Samples were put to 8% gel together with remaining lysates on Sunday


Notes

  • 50 μL of beads were used instead of 30 μL

Related

Related entries

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