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

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{|{{table}} width="800"
{|{{table}} width="800"
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Stimulation hTERT cells v2</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Co-immunoprecipitation part II, (Stimulation hTERT cells v2)</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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* 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==
===Materials===
===Materials===
*  
* RIPA buffer (per mL)
** 1.06 mg β-glycerolphosphate
** 1 mL [http://wikilaboratory.com/index.php?title=RIPA_Buffer RIPA buffer]
** 1 μL Apoprotein (1 mg/mL)
** 1 μL Leupeptin (1 mg/mL)
** 1 μL Pepstatin (A) (1 mg/mL)
** 5 μL Na<sub>3</sub>VO<sub>4</sub>
** 5 μL NaF (200 mM)
* [[PBS]]
*[http://www.piercenet.com/Products/Browse.cfm?fldID=02020105 Coomassie (Bradford) Protein Assay Kit]
** [http://www.piercenet.com/Products/Browse.cfm?fldID=02020101 BCA Protein Assay Reagent (bicinchoninic acid)]


===Method===
===Method===
====[http://genome.med.yale.edu/images/a/aa/BCA.pdf Determination of protein concentration]====
* Use cells put on S<sup>0</sup> [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/23| 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 [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/23| 23Feb2010]]
* Prepare for all donors (D9 & D12)
# Only beads (30 μL)
# Beads w. antibody (30 μL)
# Beads w. antibody (30 μL) & lysis buffer (200 μL)
# Beads w. antibody (30 μL) & sample (200 μL)
(possible also beads (30 μL) with only sample (200 μL))
* Incubate ON @ 4 °C
===Stimulation hTERT (BSc students===
* HT31P dose response curve (0 - 50 μM)
* HT31 (200 μM) /S<sup>0</sup> Basal
==Results==
{| border="1"
{| border="1"
|+'''6-wells'''
|+'''Standard curve Pierce Assay'''
!
!<u>1</u>
!<u>2</u>
!<u>3</u>
|-
!A
|
|
|
|-
!B
|
|
|
!Prot. Conc. (μg/mL)
|
!Av. Absorb. (595 nm)
|-
!Intersect
|}
!Slope
{| border="1"
!R<sup>2</sup>
|+'''24-wells'''
!
!<u>1</u>
!<u>2</u>
!<u>3</u>
!<u>4</u>
!<u>5</u>
!<u>6</u>
|-
|-
!A
!A
|
|2000
|
|0.698
|
|0.1106
|
|0.0003
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|0.993
|
|-
|-
!B
!B
|
|1500
|
|0.527
|
|
|
|
|-
|-
!C
!C
|
|1000
|
|0.421
|
|
|
|
|-
!D
|
|
|
|
|
|
|-
|}
{| border="1"
|+'''96-wells plate'''
|
!01
!02
!03
!04
!05
!06
!07
!08
!09
!10
!11
!12
|-
!A
|
|
|
|
|
|
|
|
|
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|-
!B
|
|
|
|
|
|
|
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|-
!C
|
|
|
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|
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|
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|-
|-
!D
!D
|
|750
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|0.357
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|-
|-
!E
!E
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|500
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|0.256
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|-
|-
!F
!F
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|250
|
|0.182
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|-
|-
!G
!G
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|125
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|0.153
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|-
|-
!H
!H
|0
|0.090
|-
|}
<BR><BR>
{| border="1"
|+'''Measurement concentration samples'''
|
|
!Av. Absorb. (595 nm)
!Prot. Conc. (mg/mL)
!Prot. Conc. (corrected)(mg/mL)
|-
!D9
|0.542
|1.470
|
|
|
|-
|
!D9 (1:5)
|
|0.219
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|0.368
|
|1.84
|
|-
|
!D12
|
|0.541
|
|1.466
|
|
|-
|-
!I
!D12 (1:5)
|
|0.204
|
|0.317
|
|1.58
|
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|-
|-
|}
|}


==Results==
===Related entries===
*{{todo| WHAT?}}
====Run 2: Ht31 Dose/Effect Curve & S<sup>0</sup> D9/D12====
 
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/19|Counting cells 19Feb2010]]
==Conclusion==
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/23|Putting to S<sup>0</sup> 23Feb2010]]
*{{done}} Ow..
==Related==  
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/19| Counting and splitting cells]]
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/23| Putting cells to S<sup>0</sup>, Co-immunoprecipitation]]
===Same actions===
===Same actions===
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/16| Stimulation hTERT cells]]
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/16| Stimulation hTERT cells]]


==Related topics==
* [[Cell_and_tissue_lysis_hub]]
* [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_Lysis_Buffer]]
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__NOTOC__
__NOTOC__

Revision as of 08:02, 23 March 2010

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

Co-immunoprecipitation 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 @ 4 °C

Stimulation hTERT (BSc students

  • HT31P dose response curve (0 - 50 μM)
  • HT31 (200 μM) /S0 Basal

Results

Standard curve Pierce 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)(mg/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 entries

Run 2: Ht31 Dose/Effect Curve & S0 D9/D12

Same actions

Related topics


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