User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/15: 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;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Co-IP & ELISA prep</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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==Summary==
==Summary==
* Short summary
* Co-IP
** Cell lysation
** Preparation of beads
* S<sup>0</sup> 24-wells
*Coat ELISA Plate
 


==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]]
* DMEM S<sup>0</sup>
*[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)]
* ELISA coating buffer
**1.24 g Na2CO3 in 100 mL (Buffer A)
**2.52 g NaHCO3 in 300 mL (Buffer B)
**Take 70 mL of Buffer A
**Add Buffer B until pH of 9.6 has reached (150 - 200 mL)
* IL-8 coating antibody
* 96-wells plate (MaxiSorb)


===Method===
===Method===
{| border="1"
====Putting cells to S<sup>0</sup>====
|+'''6-wells'''
* Wash cells twice with PBS
!#
* Add 500 μL DMEM S<sup>0</sup> to each well
!<u>1</u>
* Incubate @ 37 °C
!<u>2</u>
 
!<u>3</u>
====[http://genome.med.yale.edu/images/a/aa/BCA.pdf Determination of protein concentration]====
|-
* Use cells on S<sup>+</sup> refreshed [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/12| 12March2010]]
!A
** 1 Ø 10 cm dish each donor
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* Put cells on ice
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* Remove medium
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* Wash twice with 5 mL cold PBS
|-
* Add 1 mL RIPA buffer
!B
* Scrape of cells and collect in 1.5 mL tube
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* Sonicate (4x short pulse)
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* Take 10 μL of undiluted and a 5x diluted samples for Bradford assay (Pierce determination)
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* Store samples @ -20 °C
|-
====Co-immunoprecipitation part I====
|}
* Beads were coated with anti-PKA antibodies.
{| border="1"
* For <b>2</b> samples (D9/D12; Only Beads, Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample)
|+'''24-wells'''
** 280 μL beads
!#
** Spin down and remove EtOH
!<u>1</u>
** Wash with excess (400 μL) PBS
!<u>2</u>
** Spin down and remove PBS
!<u>3</u>
** Add 1:1 PBS:Beads (280 μL)
!<u>4</u>
*** 200 μL for Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample (D9/D12)
!<u>5</u>
*** 80 μL for Only Beads (D9/D12)
!<u>6</u>
*** (30 μL for each condition)
|-
** Add 1:100 (PKA RIIβ) antibody (or not for control), 2 μL
!A
** Incubate ON @ 4 °C
|
 
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===Coating of ELISA plate===
|
* Mix
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** 24 mL coating buffer (date of preparation 11March2010)
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** 80 μL primary antibody
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* Add 100 μL per well (96-wells plate)
|-
* Incubate ON
!B
 
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==Notes==
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* 6-wells plates which had their medium refreshed [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/12| 12March2010]] were thrown out
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* In putting cells to S<sup>0</sup> D12 well A2 got 1 mL instead of 0.5 mL DMEM S<sup>0</sup>, no large complications are to be expected.
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* Cells used for Co-IP and those put to S<sup>0</sup> were
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** D9 P23, plated out [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/12|12March2010]]
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** D12 P27, plated out [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/12|12March2010]]
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* For the Pierce determination new vials B and C of the standard curve were prepared.
!C
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|+'''96-wells plate'''
|#
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==Results==
==Results==
*{{todo| WHAT?}}
*Pierce determination Co-IP
** D9: 2.43 mg/mL
** D12: 2.10 mg/mL


==Conclusion==
==Conclusion==
*{{done}} Ow..
*There is enough protein present for Co-IP (>1mg/mL)




Revision as of 12:02, 15 March 2010

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

Co-IP & ELISA prep <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

  • Co-IP
    • Cell lysation
    • Preparation of beads
  • S0 24-wells
  • Coat ELISA Plate


Materials & Methods

Materials

  • RIPA buffer (per mL)
    • 1.06 mg β-glycerolphosphate
    • 1 mL RIPA buffer
    • 1 μL Apoprotein (1 mg/mL)
    • 1 μL Leupeptin (1 mg/mL)
    • 1 μL Pepstatin (A) (1 mg/mL)
    • 5 μL Na3VO4
    • 5 μL NaF (200 mM)
  • PBS
  • DMEM S0
  • Coomassie (Bradford) Protein Assay Kit
  • ELISA coating buffer
    • 1.24 g Na2CO3 in 100 mL (Buffer A)
    • 2.52 g NaHCO3 in 300 mL (Buffer B)
    • Take 70 mL of Buffer A
    • Add Buffer B until pH of 9.6 has reached (150 - 200 mL)
  • IL-8 coating antibody
  • 96-wells plate (MaxiSorb)

Method

Putting cells to S0

  • Wash cells twice with PBS
  • Add 500 μL DMEM S0 to each well
  • Incubate @ 37 °C

Determination of protein concentration

  • Use cells on S+ refreshed 12March2010
    • 1 Ø 10 cm dish each donor
  • 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)
  • Store samples @ -20 °C

Co-immunoprecipitation part I

  • Beads were coated with anti-PKA antibodies.
  • For 2 samples (D9/D12; Only Beads, Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample)
    • 280 μL beads
    • Spin down and remove EtOH
    • Wash with excess (400 μL) PBS
    • Spin down and remove PBS
    • Add 1:1 PBS:Beads (280 μL)
      • 200 μL for Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample (D9/D12)
      • 80 μL for Only Beads (D9/D12)
      • (30 μL for each condition)
    • Add 1:100 (PKA RIIβ) antibody (or not for control), 2 μL
    • Incubate ON @ 4 °C

Coating of ELISA plate

  • Mix
    • 24 mL coating buffer (date of preparation 11March2010)
    • 80 μL primary antibody
  • Add 100 μL per well (96-wells plate)
  • Incubate ON

Notes

  • 6-wells plates which had their medium refreshed 12March2010 were thrown out
  • In putting cells to S0 D12 well A2 got 1 mL instead of 0.5 mL DMEM S0, no large complications are to be expected.
  • Cells used for Co-IP and those put to S0 were
  • For the Pierce determination new vials B and C of the standard curve were prepared.

Results

  • Pierce determination Co-IP
    • D9: 2.43 mg/mL
    • D12: 2.10 mg/mL

Conclusion

  • There is enough protein present for Co-IP (>1mg/mL)



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