User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/23: Difference between revisions

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(possible also beads (30 μL) with only sample (200 μL))
(possible also beads (30 μL) with only sample (200 μL))
* Incubate ON @ °C
* Incubate ON @ °C
====hTERT stimulation====
* Remove medium form 24-wells plate with ASMC's grown ON in DMEM (S<sup>0</sup>)
* Rinse twice with warm PBS and remove buffer
* Add 200 μL DMEM S<sup>0</sup> with either HT31 or HT31P (see schedule below)
** Incubate 20 min.
* Add 400 μL DMEM S<sup>0</sup> to CTR (lane A)
* Add 200 μL DMEM S<sup>0</sup> to CSE (lane B)
* Add 200 μL 8-pctp or 6-benz according to schedule below (lane C, D)
* Wait 25 min.
** Prepare 25 mL DMEM S<sup>0</sup> with 100% CSE
** Dilute 100% CSE to 45% CSE
* Add 200 μL 45% CSE to wells according to schedule below (lane B, C D)
* Incubate 24 h.
{| border="1"
{| border="1"
|+'''6-wells'''
|+'''Stimulation set-up'''
!#
!
!<u>1</u>
!{{todo|S<sup>0</sup>}}<BR><u>1</u>
!<u>2</u>
!{{todo|S<sup>0</sup>}}<BR><u>2</u>
!<u>3</u>
!{{todo|S<sup>0</sup>}}<BR><u>3</u>
!{{done|HT31}}<BR><u>4</u>
!{{done|HT31}}<BR><u>5</u>
!{{done|HT31}}<BR><u>6</u>
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!A
|<b>A</b><BR> (CTR)
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|<b>B</b><BR> (CSE)
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|<b>C</b><BR> (CSE+8-pcpt)
{| border="1"
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|+'''24-wells'''
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!#
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{| border="1"
|+'''96-wells plate'''
|#
!01
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|<b>D</b><BR> (CSE+6-Benz)
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<b>CTR = control, CSE = Cigarette Smoke extract, 8-pcpt = 8-pcpt-2'-o-me-camp</b>


==Results==
==Results==

Revision as of 01:14, 23 March 2010

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

Project name <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

  • Cell lysis Co-IP
  • Co-IP Day II
  • Stimulation
    • ELISA
    • VASP
  • Western blot, secondary antibody
  • CHECK AVAILABILITY WESTERN BLOT THURSDAY!
  • REGISTER FOR MLII 24/3/10

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

  • resuspend beads prepared 23Feb2010
  • Prepare for all donors (D9 & D12)
  1. Only beads (30 μL)
  2. Beads w. antibody (30 μL)
  3. Beads w. antibody (30 μL) & lysis buffer (200 μL)
  4. Beads w. antibody (30 μL) & sample (200 μL)

(possible also beads (30 μL) with only sample (200 μL))

  • Incubate ON @ °C

hTERT stimulation

  • Remove medium form 24-wells plate with ASMC's grown ON in DMEM (S0)
  • Rinse twice with warm PBS and remove buffer
  • Add 200 μL DMEM S0 with either HT31 or HT31P (see schedule below)
    • Incubate 20 min.
  • Add 400 μL DMEM S0 to CTR (lane A)
  • Add 200 μL DMEM S0 to CSE (lane B)
  • Add 200 μL 8-pctp or 6-benz according to schedule below (lane C, D)
  • Wait 25 min.
    • Prepare 25 mL DMEM S0 with 100% CSE
    • Dilute 100% CSE to 45% CSE
  • Add 200 μL 45% CSE to wells according to schedule below (lane B, C D)
  • Incubate 24 h.
Stimulation set-up
S0
1 		S0
2 		S0
3 		HT31
4 		HT31
5 		HT31
6
A
(CTR)
B
(CSE)
C
(CSE+8-pcpt)
D
(CSE+6-Benz)

CTR = control, CSE = Cigarette Smoke extract, 8-pcpt = 8-pcpt-2'-o-me-camp

Results

  • WHAT?

Conclusion

  • DONE Ow..

Related

Related entries

Same actions



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