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

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==Summary==
==Summary==
===SDS-PAGE===
===Western blot===
*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
|-
|}
* Transfer to blot
* Block blot ON in 1% BSA in TBS-T
===Washing beads: cAMP precipitation===
===Washing beads: cAMP precipitation===
*Spin beads 5 min. 2100xg @ 4 °C
*Spin beads 5 min. 2100xg @ 4 °C
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==Notes==
==Notes==
*One gel was too small, probably added water to fast (to remove airbubbles from top). I only needed 5 so it's okay.
*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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*Second wash of beads was kept in centrifuge for ~30 min. and centrifuged a second time before continuing to third wash
*Third wash was performed with new RIPA buffer
*Gel running was troublesome, first a bit blurry but straightened out later. Marker was a bit faint and running took >2 h.
**HiMark (Invitrogen) was used
* Upper 3 bands were not completely transferred to the membrane
 
==Results==
==Results==
*{{todo| WHAT?}}
*Ponceau staining of cAMP precipitations 4 gels with the same samples and marker
[[Image:22062010_Ponceau_staining_cAMP_precipitation.png|500px]]


==Discussion==
==Discussion==
*{{done}} Ow..
*Nothing visible, HOPEFULLY this is due to a low amount of protein (or bad Ponceau) and NOT due to the complete lack of protein
 
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==Related==
==Related==
===Related entries===
===Related entries===

Revision as of 09:42, 22 June 2010

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Summary

Western blot

  • 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
  • Transfer to blot
  • Block blot ON in 1% BSA in TBS-T

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.
  • Second wash of beads was kept in centrifuge for ~30 min. and centrifuged a second time before continuing to third wash
  • Third wash was performed with new RIPA buffer
  • Gel running was troublesome, first a bit blurry but straightened out later. Marker was a bit faint and running took >2 h.
    • HiMark (Invitrogen) was used
  • Upper 3 bands were not completely transferred to the membrane

Results

  • Ponceau staining of cAMP precipitations 4 gels with the same samples and marker

Discussion

  • Nothing visible, HOPEFULLY this is due to a low amount of protein (or bad Ponceau) and NOT due to the complete lack of protein


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