User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/15: Difference between revisions
From OpenWetWare
m (→Results) |
m (→Conclusion) |
||
(9 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;"> | |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> </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> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | ||
|- | |- | ||
Line 23: | Line 23: | ||
** 1.06 mg β-glycerolphosphate | ** 1.06 mg β-glycerolphosphate | ||
** 1 mL [http://wikilaboratory.com/index.php?title=RIPA_Buffer RIPA buffer] | ** 1 mL [http://wikilaboratory.com/index.php?title=RIPA_Buffer RIPA buffer] | ||
** 1 μL Apoprotein | ** 1 μL Apoprotein (1 mg/mL) | ||
** 1 μL Leupeptin | ** 1 μL Leupeptin (1 mg/mL) | ||
** 1 μL Pepstatin (A) | ** 1 μL Pepstatin (A) (1 mg/mL) | ||
** 5 μL Na<sub>3</sub>VO<sub>4</sub> | ** 5 μL Na<sub>3</sub>VO<sub>4</sub> | ||
** 5 μL NaF | ** 5 μL NaF (200 mM) | ||
* [[PBS]] | * [[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=02020105 Coomassie (Bradford) Protein Assay Kit] | ||
** [http://www.piercenet.com/Products/Browse.cfm?fldID=02020101 BCA Protein Assay Reagent (bicinchoninic acid)] | ** [http://www.piercenet.com/Products/Browse.cfm?fldID=02020101 BCA Protein Assay Reagent (bicinchoninic acid)] | ||
Line 36: | Line 37: | ||
**Take 70 mL of Buffer A | **Take 70 mL of Buffer A | ||
**Add Buffer B until pH of 9.6 has reached (150 - 200 mL) | **Add Buffer B until pH of 9.6 has reached (150 - 200 mL) | ||
* IL-8 coating antibody | |||
* 96-wells plate (MaxiSorb) | |||
===Method=== | ===Method=== | ||
====Putting cells to S<sup>0</sup>==== | |||
* Wash cells twice with PBS | |||
* Add 500 μL DMEM S<sup>0</sup> to each well | |||
* Incubate @ 37 °C | |||
====[http://genome.med.yale.edu/images/a/aa/BCA.pdf Determination of protein concentration]==== | ====[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]] | * Use cells on S<sup>+</sup> refreshed [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/12| 12March2010]] | ||
** 1 Ø 10 cm dish each donor | |||
* Put cells on ice | * Put cells on ice | ||
* Remove medium | * Remove medium | ||
Line 47: | Line 56: | ||
* Sonicate (4x short pulse) | * Sonicate (4x short pulse) | ||
* Take 10 μL of undiluted and a 5x diluted samples for Bradford assay (Pierce determination) | * Take 10 μL of undiluted and a 5x diluted samples for Bradford assay (Pierce determination) | ||
* Store samples @ -20 °C | |||
====Co-immunoprecipitation part I==== | ====Co-immunoprecipitation part I==== | ||
* Beads were coated with anti-PKA antibodies. | * Beads were coated with anti-PKA antibodies. | ||
* For <b> | * For <b>2</b> 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 | ** Spin down and remove EtOH | ||
** Wash with excess (400 μL) PBS | ** Wash with excess (400 μL) PBS | ||
** Spin down and remove PBS | ** Spin down and remove PBS | ||
** Add | ** Add 1:1 PBS:Beads (280 μL) | ||
** Add 1 | *** 200 μL for Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample (D9/D12) | ||
** Incubate | *** 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=== | ===Coating of ELISA plate=== | ||
* Mix | * Mix | ||
** 24 mL coating buffer | ** 24 mL coating buffer (date of preparation 11March2010) | ||
** 80 μL primary antibody | ** 80 μL primary antibody | ||
* Add 100 μL per well (96-wells plate) | * Add 100 μL per well (96-wells plate) | ||
* Incubate ON | * Incubate ON | ||
==Notes== | ==Notes== | ||
* 6-wells plates which had their medium refreshed [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/12| 12March2010]] were thrown out | |||
* 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. | |||
* Cells used for Co-IP and those put to S<sup>0</sup> were | |||
** D9 P23, plated out [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/12|12March2010]] | |||
** D12 P27, plated out [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/12|12March2010]] | |||
* For the Pierce determination new vials B and C of the standard curve were prepared. | |||
==Results== | ==Results== | ||
* | *Pierce determination Co-IP | ||
** D9: 2.43 mg/mL | |||
** D12: 2.10 mg/mL | |||
==Conclusion== | ==Conclusion== | ||
* | *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> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
Summary
Materials & MethodsMaterials
MethodPutting cells to S0
Determination of protein concentration
Co-immunoprecipitation part I
Coating of ELISA plate
Notes
Results
Conclusion
|