User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/25: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
mNo edit summary
Line 36: Line 36:
* Remove medium
* Remove medium
* Wash with PBS twice and remove
* Wash with PBS twice and remove
* Add 600 μL S<sup>0</sup> or S<sup>0</sup> 45% CSE
* Add 600 μL S<sup>0</sup> or S<sup>0</sup>+Ht31 (see hTERT VASP schedule below)
* Incubate
* Incubate 15-20 min. RT
* Add 600 μL S<sup>0</sup> or S<sup>0</sup>+6-Bnz (see hTERT VASP schedule below)
* Incubate 10 min. RT
* Add 600 μL S<sup>0</sup> or S<sup>0</sup> 45% CSE (see hTERT VASP schedule below)
* Incubate 10 min. RT
* Remove medium
* Wash with PBS and remove
* Add 200 μL Hot Lysis buffer and scrape cells with tip
* Transfer cell lysate into 1.5 mL tubes
* Store @ -20 °C
 
{| border="1"
{| border="1"
|+'''hTERT VASP'''
|+'''hTERT VASP'''
Line 56: Line 66:
|-
|-
|}
|}


==Results==
==Results==

Revision as of 07:55, 25 February 2010

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

Co-immunoprecipitation part III <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

The samples have been incubated with the (PKA) antibody coated beads and today the beads are washed and stored in Laemli buffer

Materials & Methods

Materials

  • Laemli buffer
  • PBS
  • syringe


Method

Co-immunoprecipitation

  • Centrifuge sample incubated with beads, 5 min. 14.000 RPM
  • Remove supernatant* (w. syringe)
  • Add 1 mL PBS
  • Centrifuge, 5 min. 14.000 RPM
  • Remove supernatant*
  • Add 75 μL Laemli buffer
  • Store samples @ -20 °C
    • Alternatively go directly on to Western blot (Boil samples and spin beads down, bring supernatant onto gel)


* Be careful not to touch beads

VASP

  • Use cells grown by Bachelor students TODO
  • Remove medium
  • Wash with PBS twice and remove
  • Add 600 μL S0 or S0+Ht31 (see hTERT VASP schedule below)
  • Incubate 15-20 min. RT
  • Add 600 μL S0 or S0+6-Bnz (see hTERT VASP schedule below)
  • Incubate 10 min. RT
  • Add 600 μL S0 or S0 45% CSE (see hTERT VASP schedule below)
  • Incubate 10 min. RT
  • Remove medium
  • Wash with PBS and remove
  • Add 200 μL Hot Lysis buffer and scrape cells with tip
  • Transfer cell lysate into 1.5 mL tubes
  • Store @ -20 °C
hTERT VASP
#
1
2
3
A
Control
Ht31 (50 μM)
CSE (15%)
B
CSE (15%) + Ht31 (50 μM)
CSE (15%) + 6-Bnz (500 μM)
CSE (15%) + 6-Bnz (500 μM)+ Ht31 (50 μM)

Results

  • WHAT?

Conclusion

  • DONE Ow..

Related topics



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/25&oldid=394203"