User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/24: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
mNo edit summary
mNo edit summary
Line 18: Line 18:
* 8-pcpt
* 8-pcpt
* 6-Bnz
* 6-Bnz
* CSE + Rp-8-CPT-cAMPS
 
* Rp-8-CPT-cAMPS
With a dose response range of 0 - 50 μg/mLug Ht31, instead of HT31P S<sup>0</sup> was used.
* CSE + siRNA
* siRNA


==Materials & Methods==
==Materials & Methods==
Line 266: Line 264:
|-
|-
|}
|}
 
-->
==Results==
==Results==
*{{todo| WHAT?}}
*{{todo| WHAT?}}
Line 279: Line 277:
==Related topics==
==Related topics==
* [[Cell_and_tissue_lysis_hub]]
* [[Cell_and_tissue_lysis_hub]]
* [[Protocol_Total_Protein_Isolation_using_RIPA_buffer.html]]
* [http://jacobs.openwetware.org/Protocol_Total_Protein_Isolation_using_RIPA_buffer.html Protocol_Total_Protein_Isolation_using_RIPA_buffer.html]
* [[Jacobs:Protocol_Total_Protein_Isolation_using_RIPA_buffer_2]]
* [[Jacobs:Protocol_Total_Protein_Isolation_using_RIPA_buffer_2]]
* [[Jacobs:Protocol_Total_Protein_Isolation_Using_RIPA_Lysis_Buffer]]
* [[Jacobs:Protocol_Total_Protein_Isolation_Using_RIPA_Lysis_Buffer]]

Revision as of 05:00, 24 February 2010

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

Co-stimulation part II, (Stimulation hTERT cells v2) <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

Conditions

  • CTR
  • CSE
  • CSE + 8-pcpt
  • CSE + 6-Bzn
  • 8-pcpt
  • 6-Bnz

With a dose response range of 0 - 50 μg/mLug Ht31, instead of HT31P S0 was used.

Materials & Methods

Materials

Method

Determination of protein concentration

  • Use cells put on S0 23Feb2010
  • Put cells on ice
  • Remove medium
  • Wash twice with 5 mL cold PBS
  • Add 1 mL RIPA buffer
  • Scrape of cells and collect in 1.5 mL tube
  • Sonicate (4x short pulse)
  • Take 10 μL of undiluted and a 5x diluted samples for Bradford assay (Pierce determination)

Co-immunoprecipitation

  • Spin down beads prepared 23Feb2010
    • Remove liquid
  • Add PBS to wash
  • Spin down and remove liquid
  • Prepare
  1. Only beads (100 μL)
  2. Beads w. antibody (100 μL)
  3. Beads w. antibody (100 μL) & lysis buffer (200 μL)
  4. Beads w. antibody (100 μL) & sample (200 μL)

(possible also beads (100 μL) with only sample (200 μL))

  • Incubate?
  • Wash?
  • Add 75 μL 4x loading buffer
  • Boil samples and put on gel

Results

  • WHAT?

Conclusion

  • DONE Ow..

Related

Same actions

Related topics