User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/22

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Co-IP & Western blot Main project page
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Summary

  • Co-IP Day I
  • Western blot Co-IP
  • Put cells to S0

Materials & Methods

Method

Co-immunoprecipitation part I

  • Beads were coated with anti-PKA antibodies.
  • For 2 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
    • Wash with excess (400 μL) PBS
    • Spin down and remove PBS
    • Add 1:1 PBS:Beads (280 μL)
      • 200 μL for Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample (D9/D12)
      • 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

SDS-PAGE

  • Clean the glass plates (UP, Soap, UP, EtOH and dry)
  • Prepare elaboration, look out for leakage!
  • Choose % of gel, big proteins require higher % (here 10% SDS gel)
  • Prepare separating gel (pour until green beam, ~10 mL per gel)
    • Add UP until flooded
  • When polymerized (approx. 30 min) remove isolating layer (remove UP with tissue paper)
  • Prepare 5% stacking gel
  • Pour until flooding and add combs
    • Dilute samples with the buffer which they are stored in until an equal amount is reached (20 ng in 30 μL)
    • Add 10 μL 4x loading buffer
    • Boil samples for 5 min. and cool samples on ice
  • Bring gels into the electrophoresis elaboration and pour ELFO buffer in the inner space to check for leakage
  • Remove combs and rinse slots with buffer
  • Fill slots with sample (40 μL) and marker (15 μL)
  • Start electrophoresis (40 min. 200 V)
    • Alternatively start with 100 V until samples reach the separating gel and increase voltage

Western Blot

  • Moisten sponges, filter papers and membrane in transfer buffer
  • Prepare sandwich
    • Black clamp
    • Sponge
    • Whatman paper
    • Gel
    • Membrane
    • Whatman paper
    • Sponge
    • White clamp
  • Put sandwhich in the transferring system (black to black) and fill it with transferbuffer
  • Transfer proteins to membrane for 1 hour @ 100 V on ice while stirring
  • After transfer check if marker is visible on membrane (successful transfer)
    • Alternatively Ponceau S staining is possible (not used @ this lab)
    • Remove LEFT UPPER corner to mark what is what
  • Block membrane with 5% milk solution in TBST buffer (ON, 4°C)
    • Alternatively 2 h @ RT
  • Treat with primary antibody (diluted in 5% TBST-milk solution) (ON, 4°C)
    • GAPDH 1:2000
    • VASP 1:1000
    • AKAP 1:1000
  • Wash 3x 10 min. in TBST buffer (first can be less)
  • Treat with secondary antibody (diluted in 5% TBST-milk solution) (1h, RT)
  • Wash 3x 10 min. in TBST buffer
Gel loading
# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Gel 1 Marker
(15 μL)
D9A 1
(all <30 μL)
D9A 2
(all <30 μL)
D9A 3
(all <30 μL)
D9A 4
(all <30 μL)
D12A 1
(30 μL)
D12A 2
(30 μL )
D12A 3
(30 μL)
D12A 4
(30 μL)
D9B 1
(30 μL)
D9B 2
(30 μL)
D9B 3
(30 μL)
D9B 4
(30 μL)
Gel 2 Marker
(15 μL)
D12B 1
(35 μL)
D12B 2
(35 μL)
D12B 3
(35 μL)
D12B 4
(35 μL)
X D9C 1
(35 μL)
D9C 2
(35 μL)
D9C 3
(35 μL)
D9C 4
(35 μL)
D12C 1
(35 μL)
D12C 2
(35 μL)
D12C 3
(35 μL)
D12C 4
(35 μL)
  1. Only beads (30 μL)
  2. Beads w. antibody (30 μL)
  3. Beads w. antibody (30 μL) & lysis buffer (200 μL)
  4. Beads w. antibody (30 μL) & sample (200 μL)

A. 23Feb2010 to 25Feb2010
B. 03March2010 to 05March2010
C. 15March2010 to 17March2010

Notes

  • No ice block was put in transfer system for the first 24 min. (of 60 min. in total)
  • Membrane was blocked for 1.5 h @ RT (14.22 - 15.52)

Related

Related entries

A. 23Feb2010 to 25Feb2010
B. 03March2010 to 05March2010
C. 15March2010 to 17March2010

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