User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/06/22: Difference between revisions

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===SDS-PAGE===
===SDS-PAGE===
*Prepare 6x 8% gels
*Prepare 6x 8% gels
*Load 4 gels
{| border="1"
|+'''Gel loading hTERT D9 + HEK293 see codes [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/06/21|21June2010]]'''
! align="center" style="background:#f0f0f0;"|#
! align="center" style="background:#f0f0f0;"|<u>1</u>
! align="center" style="background:#f0f0f0;"|<u>2</u>
! align="center" style="background:#f0f0f0;"|<u>3</u>
! align="center" style="background:#f0f0f0;"|<u>4</u>
! align="center" style="background:#f0f0f0;"|<u>5</u>
! align="center" style="background:#f0f0f0;"|<u>6</u>
! align="center" style="background:#f0f0f0;"|<u>7</u>
! align="center" style="background:#f0f0f0;"|<u>8</u>
! align="center" style="background:#f0f0f0;"|<u>9</u>
! align="center" style="background:#f0f0f0;"|<u>10</u>
|-
!Gel<br>cAMP prec.
| align="center"|X
| align="center"|HEK293<br>(20 μL)
| align="center"|[[Media:HiMark_protein_marker_-_Invitrogen.jpg|Marker]]<br> (8 μL)
| align="center"|C1<br>(20 μL)
| align="center"|C2<br>(20 μL)
| align="center"|C3<br>(20 μL)   
| align="center"|S1<br>(20 μL)
| align="center"|S2<br>(20 μL)
| align="center"|S3<br>(20 μL)
| align="center"|X
|-
|}
===Washing beads: cAMP precipitation===
===Washing beads: cAMP precipitation===
*Spin beads 5 min. 2100xg @ 4 °C
*Spin beads 5 min. 2100xg @ 4 °C

Revision as of 06:52, 22 June 2010

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

cAMP precipitation <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

SDS-PAGE

  • Prepare 6x 8% gels
  • Load 4 gels
Gel loading hTERT D9 + HEK293 see codes 21June2010
# 1 2 3 4 5 6 7 8 9 10
Gel
cAMP prec. 		X HEK293
(20 μL) 		Marker
(8 μL) 		C1
(20 μL) 		C2
(20 μL) 		C3
(20 μL) 		S1
(20 μL) 		S2
(20 μL) 		S3
(20 μL) 		X

Washing beads: cAMP precipitation

  • Spin beads 5 min. 2100xg @ 4 °C
  • Collect supernatant (= NAC)
  • Add 500 μL Potter buffer
  • Spin 5 min. 2100xg @ 4 °C
  • Remove supernatant with vacuum pump
  • Add 500 μL Potter buffer
  • Spin 5 min. 2100xg @ 4 °C
  • Remove supernatant with vacuum pump
  • Add 500 μL Potter buffer
  • Spin 5 min. 2100xg @ 4 °C
  • Remove supernatant with vacuum pump and syringe
  • Add 50 μL of 4x sample buffer
  • Spin 5 min. 2100xg @ 4 °C

RIPA BUFFER (100 mL)

  • 50 mM Tris-HCl (5 mL 1 M Tris pH 7.4)
  • Adjust to pH 7.4
  • 150 mM NaCl (0.87 g NaCl)
  • 1 mM EDTA (37.22 mg EDTA)
  • 1% Triton x-100 (1 mL Tritox x-100)
  • 1% Sodium deoxycholate (1,03 g (97%))
  • 0.1% SDS (1 g SDS)

Notes

  • One gel was too small, probably added water to fast (to remove airbubbles from top). I only needed 5 so it's okay.


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