User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/05/26

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New cells, cAMP-precipitation, AKAP79 immunoblot of cAMP-precipitation Main project page
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Summary

The current cell line running is in passage 28 and therefore a new one will be started from the freezer. To see what AKAPs are seen in the RII overlay of 19May2010 immunoblots will require the same samples to be put to gel again. The ponceau staining appears to show the same results seen 09May2010. Due to the focus of Groningen on AKAP79, so far the only AKAP confirmed in this cell line, the first antibody will be targetted to this AKAP.

Materials & Methods

Materials

Method

Plating cells from -80 °C D9 P23

  • Take a Ø10cm dish.
  • Put 10 mL of medium in a 15 mL Falcon tube.
  • Get the cells out of the -80°C.
  • Defrost the cells quickly by using a water bath.
  • As soon as the cells are defrosted, pipette the cells into the medium which is in the Falcon tube.
  • Put the medium containing the cells into the petridish.
  • Put O.N. at 37°C

Putting samples to gel for SDS-PAGE

  • Put samples of 7.5.2010 onto gel prepared 6.5.2010 stored @ 4 °C
Biorad Precision Plus Protein Dual Color marker
  • Add 8 μL of marker and 20 μL of samples
    • Boil samples for 5 min. and let samples cool
  • Bring gels into the electrophoresis elaboration and pour buffer in the inner space
  • Remove combs and rinse slots with buffer
  • Fill slots with sample (20 μL) and Precision Plus Protein Dual Color marker (8 μL)
  • Start electrophoresis (60 min. 100 V)
# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
SampleMarkerCSE beadsCSE NACCSE LysateCSE (-) beadsCSE (-) NACCSE (-) LysatemarkerS0 beadsS0 NACS0 LysateS0 (-) beadsS0 (-) NACS0 (-) Lysatex

Dry Western Blot

  • Put two filter papers on the dry blot apparatus (filters moistened in transfer buffer)
  • Put on the membrane
    • Rinse membrane first 15 sec. with 100% EtOH
    • Rinse with dH2O
    • Moisten with transfer buffer (2 min.)
  • Put on the gel
  • Finish with two moistend filter papers and close apparatus
  • Run for 90 min. @ 10 V (75 mA per gel should been seen)

Notes

  • Gel was actually too old to use, (-) samples were all a bit lower than the other samples
  • (-) samples put to beads treated cAMP first
  • NAC = sample taken from beads after ON incubation of sample with beads

Immunoblot

  • Membrane was treated with Ponceau S staining and blocked in 1% BSA TBST solution for >1 h @ RT
  • Incubate with primary antibody ON @ 4 °C
    • Antibodies used on membrane
    • Mouse monoclonal antibody - AKAP79 (D9): sc-17772 Santa Cruz Biotechnology, Inc. (200 μg/mL)
    • 1:1000 dilution in blocking solution
  • Wash 3x 10 min. with blocking solution
  • Incubate with secondary antibody 1 h @ RT
    • anti-mouse
    • 1:10.000 dilution in blocking solution
  • Wash 3x 10 min. with blocking solution

2x new 8% polyacrylamide gels were prepared

  • 15 wells combs
  • In bright sun light, maybe heat is not a good thing

cAMP precipitation & Preparations RII overlay

  • Cells lysed on 07May2010 were used again
  • 900 μL was put to 30 μL cAMP coated agarose beads and incubated @ 4 °C ON while mixing
  • 75 μL was mixed with 25 μL 4x sample buffer and incubated @ 95 °C for 5 min.
    • Samples were put to -20 °C together with remaining lysate

Results

  • Ponceau staining

Discussion

  • The ponceau staining appears to show the same results seen 09May2010.

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