User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/08/13: Difference between revisions

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* Samples were put to 70 °C for 10 min.
* Samples were put to 70 °C for 10 min.
* Samples were put to 4-12% Gradient NuPage gel 200 V
* Samples were put to 4-12% Gradient NuPage gel 200 V
{| border="1"
|+'''4-12% Gradient NuPage Gel loading LYSATES hTERT D9 + HEK293, HeLa -S & Jurkat 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>
! align="center" style="background:#f0f0f0;"|<u>11</u>
! align="center" style="background:#f0f0f0;"|<u>12</u>
|-
!Gel<br>cAMP prec.
| align="center"|[[Media:HiMark_protein_marker_-_Invitrogen.jpg|Invitrogen Marker]]<br> (8 μL)
| align="center"|Jurkat <br>(20 μL)
| align="center"|HeLa -S <br>(20 μL)
| align="center"|HEK293<br>(20 μL)
| align="center"|[[Media:Biorad_-_Precision_Plus_Protein_Dual_Color_marker.jpg|Biorad 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
|-
|}


===Stripping blot===
===Stripping blot===

Revision as of 02:34, 13 August 2010

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Summary

  • TODO Strip blot


Materials & Methods

SDS-PAGE

  • Lysate samples were prepared, undiluted samples (75 µL) with NuPage loading buffer + DTT (25 µL)
  • Samples were put to 70 °C for 10 min.
  • Samples were put to 4-12% Gradient NuPage gel 200 V
4-12% Gradient NuPage Gel loading LYSATES hTERT D9 + HEK293, HeLa -S & Jurkat see codes 21June2010
# 1 2 3 4 5 6 7 8 9 10 11 12
Gel
cAMP prec. 		Invitrogen Marker
(8 μL) 		Jurkat
(20 μL) 		HeLa -S
(20 μL) 		HEK293
(20 μL) 		Biorad Marker
(8 μL) 		C1
(20 μL) 		C2
(20 μL) 		C3
(20 μL) 		S1
(20 μL) 		S2
(20 μL) 		S3
(20 μL) 		X

Stripping blot

  • Stripping solution
    • 50 mL dH2O
    • 5 mL Tris-Cl (1.0 M, pH 6.8) (Stacking gel buffer SDS-PAGE)
    • 1 g SDS
    • 150 mg DTT (Add later)
  1. Prewarm buffer (w/o DTT) for 30 min. to 70 °C in waterbath
  2. Add DTT just before adding membrane
  3. Completely immerse membrane
  4. Incubate 30 min @ 70 °C
  5. Wash 3x with dH2O or TBS-T
  6. Membrane can now be blocked again


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