User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/05/28: Difference between revisions

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====SDS-PAGE====
====SDS-PAGE====
* HEK lysate, hTERT RIPA & Potter lysate & cAMP beads/NAC
* HEK lysate, hTERT RIPA & Potter lysate & cAMP beads/NAC
* Put samples to 8% polyacrylamide gel
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''#'''
| align="center" style="background:#f0f0f0;"|'''1'''
| align="center" style="background:#f0f0f0;"|'''2'''
| align="center" style="background:#f0f0f0;"|'''3'''
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| align="center" style="background:#f0f0f0;"|'''6'''
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| align="center" style="background:#f0f0f0;"|'''8'''
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| align="center" style="background:#f0f0f0;"|'''14'''
| align="center" style="background:#f0f0f0;"|'''15'''
|-
| Sample||Marker||CSE beads||CSE NAC||CSE Lysate Potter||CSE Lysate RIPA||x||HEK293 RIPA lysate||marker||S<sup>0</sup> beads||S<sup>0</sup> NAC||S<sup>0</sup> Lysate Potter||S<sup>0</sup> Lysate RIPA||x||x||x
|-
|}


==Materials & Methods==
==Materials & Methods==

Revision as of 01:53, 28 May 2010

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Summary

Immunoblot

  • Wash blot incubated ON 3x 10 min. with blocking solution
  • Incubate with secondary antibody 1 h @ RT
    • anti-rabbit
    • 1:10.000 dilution in blocking solution
  • Wash 3x 10 min. with blocking solution

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 plastic
  • Take picture (depends on what is available)

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

SDS-PAGE

  • HEK lysate, hTERT RIPA & Potter lysate & cAMP beads/NAC
  • Put samples to 8% polyacrylamide gel
# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sample Marker CSE beads CSE NAC CSE Lysate Potter CSE Lysate RIPA x HEK293 RIPA lysate marker S0 beads S0 NAC S0 Lysate Potter S0 Lysate RIPA x x x

Materials & Methods

Materials

Method

6-wells
# 1 2 3
A
B
24-wells
# 1 2 3 4 5 6
A
B
C
D
96-wells plate
# 01 02 03 04 05 06 07 08 09 10 11 12
A
B
C
D
E
F
G
H
I

Notes

Results

  • WHAT?

Discussion

  • DONE Ow..

Related

Related entries

Same actions

Related topics

Attachment

<html> <a href="http://openwetware.org/wiki/Image:HTERT_Donor_12_ht31_160310.xls"> 

  <img src="http://openwetware.org/images/f/f8/Owwnotebook_icon.png" alt="" width="50" height="50">

</a> </html> Excel TITLE


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