User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/24: 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
<!--{| border="1"
 
|+'''6-wells'''
==Results==
!
!<u>1</u>
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!A
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!B
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{| border="1"
{| border="1"
|+'''24-wells'''
|+'''Standard curve Bradford Assay'''
!
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!<u>6</u>
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!B
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!C
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!Prot. Conc. (μg/mL)
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!Av. Absorb. (595 nm)
!D
!Intersect
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!Slope
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!R<sup>2</sup>
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{| border="1"
|+'''96-wells plate'''
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!01
!02
!03
!04
!05
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!09
!10
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!12
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!A
!A
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|2000
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|0.698
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|0.1106
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|0.0003
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|0.993
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!B
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|1500
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|0.527
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!C
!C
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|1000
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!D
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!E
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|500
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!F
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|250
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|0.182
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!G
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|125
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!H
!H
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|0.090
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<BR><BR>
{| border="1"
|+'''Measurement concentration samples'''
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!Av. Absorb. (595 nm)
!Prot. Conc. (mg/mL)
!Prot. Conc. (corrected)(μg/mL)
|-
!D9
|0.542
|1.470
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!D9 (1:5)
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|0.219
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|0.368
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|1.84
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!D12
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|0.541
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|1.466
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!I
!D12 (1:5)
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|0.204
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|0.317
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|1.58
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-->
==Results==
*{{todo| WHAT?}}
==Conclusion==
*{{done}} Ow..
==Related==  
==Related==  
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/19|  Counting and splitting cells]]
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/19|  Counting and splitting cells]]

Revision as of 05:50, 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

  • 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

Results

Standard curve Bradford Assay
Prot. Conc. (μg/mL) Av. Absorb. (595 nm) Intersect Slope R2
A 2000 0.698 0.1106 0.0003 0.993
B 1500 0.527
C 1000 0.421
D 750 0.357
E 500 0.256
F 250 0.182
G 125 0.153
H 0 0.090



Measurement concentration samples
Av. Absorb. (595 nm) Prot. Conc. (mg/mL) Prot. Conc. (corrected)(μg/mL)
D9 0.542 1.470
D9 (1:5) 0.219 0.368 1.84
D12 0.541 1.466
D12 (1:5) 0.204 0.317 1.58

Related

Same actions

Related topics


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