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

From OpenWetWare
Jump to navigationJump to search
(14 intermediate revisions by the same user not shown)
Line 5: Line 5:
{|{{table}} width="800"
{|{{table}} width="800"
|-
|-
|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 Part II, hTERT Stimulation & Western blot cont'd</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>}}
|-
|-
Line 15: Line 15:
* Stimulation  
* Stimulation  
** ELISA
** ELISA
** VASP
** <del>VASP</del>
* Western blot, secondary antibody
* Western blot, secondary antibody
* CHECK AVAILABILITY WESTERN BLOT THURSDAY!
* REGISTER FOR MLII 24/3/10


==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)
# Beads (30 μL) with only sample (200 μL))
* Incubate ON @ 4 °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 S<sup>0</sup> (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>
|-
|-
!A
|<b>A</b><BR> (CTR)
|
|style="background:red"|
|
|style="background:red"|
|
|style="background:red"|
|style="background:green"|
|style="background:green"|
|style="background:green"|
|-
|-
!B
|<b>B</b><BR> (CSE)
|
|style="background:red"|
|
|style="background:red"|
|
|style="background:red"|
|style="background:green"|
|style="background:green"|
|style="background:green"|
|-
|-
|}
|<b>C</b><BR> (CSE+8-pcpt)
{| border="1"
|style="background:red"|
|+'''24-wells'''
|style="background:red"|
!#
|style="background:red"|
!<u>1</u>
|style="background:green"|
!<u>2</u>
|style="background:green"|
!<u>3</u>
|style="background:green"|
!<u>4</u>
!<u>5</u>
!<u>6</u>
|-
|-
!A
|<b>D</b><BR> (CSE+6-Benz)
|
|style="background:red"|
|
|style="background:red"|
|
|style="background:red"|
|
|style="background:green"|
|
|style="background:green"|
|
|style="background:green"|
|-
!B
|
|
|
|
|
|
|-
!C
|
|
|
|
|
|
|-
!D  
|
|
|
|
|
|
|-
|}
{| border="1"
|+'''96-wells plate'''
|#
!01
!02
!03
!04
!05
!06
!07
!08
!09
!10
!11
!12
|-
!A
|
|
|
|
|
|
|
|
|
|
|
|
|-
!B
|
|
|
|
|
|
|
|
|
|
|
|
|-
!C
|
|
|
|
|
|
|
|
|
|
|
|
|-
!D
|
|
|
|
|
|
|
|
|
|
|
|
|-
!E
|
|
|
|
|
|
|
|
|
|
|
|
|-
!F
|
|
|
|
|
|
|
|
|
|
|
|
|-
!G
|
|
|
|
|
|
|
|
|
|
|
|
|-
!H
|
|
|
|
|
|
|
|
|
|
|
|
|-
!I
|
|
|
|
|
|
|
|
|
|
|
|
|-
|-
|}
|}
<b>CTR = control, CSE = Cigarette Smoke extract, 8-pcpt = 8-pcpt-2'-o-me-camp</b>
===Western blot, secondary Antibody===
* Wash 3x 10 min. in TBST buffer (first can be less)
* Treat with secondary antibody (anti-Mouse) (diluted in 5% TBST-milk solution) (1.5h, RT)
* Wash 3x 10 min. in TBST buffer
====Preparing film====
* 1:1 ratio [[wikipedia:Chemiluminescence#Enhanced_chemiluminescence|ECL]] solutions (2 mL per membrane)
* Incubate membrane in ECL solution 5 min, RT (both sides of membrane)
* Place blot in between two overhead sheets
* Take picture (depends on what is available)
==Notes==
* 6-wells put to S<sup>0</sup> [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/22|22March2010]] were passed on to Anouk to continue --> VASP experiment cancelled due to time limitation
* For Pierce determination new standard was made, <u>only</u> concentration 750, 500 & 250 μg/mL


==Results==
==Results==
*{{todo| WHAT?}}
*Co-IP Blots of samples pulled down using PKA antibody coated beads, blots were treated with anti-AKAP antibody
 
[[Image:230310_CoIP_AKAP_Gel_1.JPG|500px]]<br>
Gel 1<br>
[[Image:230310_CoIP_AKAP_Gel_2.JPG|500px]]<br>
Gel 2<br>
# 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)
 
A. [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/23|23Feb2010]] to [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/23|25Feb2010]]<br>
B. [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/03|03March2010]] to [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/05|05March2010]]<br>
C. [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/15|15March2010]] to [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/17|17March2010]]


==Conclusion==
==Conclusion==
*{{done}} Ow..
* All Co-IP samples incubated with sample (and antibody) showed a double band it is unclear wether or not it is at the right height. The used antibody was directed against AKAP179, 86 kDa
 
==Related==
===Related entries===
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/22|Co-IP part I & Western blot]]
===Same actions===
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/24| Cell lysis & Co-IP Day II]]
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/16| Cell stimulation]]




Revision as of 09:08, 23 March 2010

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

Co-IP Part II, hTERT Stimulation & Western blot cont'd <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

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)
  5. Beads (30 μL) with only sample (200 μL))
  • Incubate ON @ 4 °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 S0 (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

Western blot, secondary Antibody

  • Wash 3x 10 min. in TBST buffer (first can be less)
  • Treat with secondary antibody (anti-Mouse) (diluted in 5% TBST-milk solution) (1.5h, RT)
  • Wash 3x 10 min. in TBST buffer

Preparing film

  • 1:1 ratio ECL solutions (2 mL per membrane)
  • Incubate membrane in ECL solution 5 min, RT (both sides of membrane)
  • Place blot in between two overhead sheets
  • Take picture (depends on what is available)

Notes

  • 6-wells put to S0 22March2010 were passed on to Anouk to continue --> VASP experiment cancelled due to time limitation
  • For Pierce determination new standard was made, only concentration 750, 500 & 250 μg/mL

Results

  • Co-IP Blots of samples pulled down using PKA antibody coated beads, blots were treated with anti-AKAP antibody


Gel 1

Gel 2

  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)

A. 23Feb2010 to 25Feb2010
B. 03March2010 to 05March2010
C. 15March2010 to 17March2010

Conclusion

  • All Co-IP samples incubated with sample (and antibody) showed a double band it is unclear wether or not it is at the right height. The used antibody was directed against AKAP179, 86 kDa

Related

Related entries

Same actions



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/23&oldid=401462"