User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/23

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Co-IP Part II, hTERT Stimulation & Western blot cont'd
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Summary

 * Cell lysis Co-IP
 * Co-IP Day II
 * Stimulation
 * ELISA
 * VASP
 * Western blot, secondary antibody

Materials

 * RIPA buffer (per mL)
 * 1.06 mg β-glycerolphosphate
 * 1 mL RIPA buffer
 * 1 μL Apoprotein (1 mg/mL)
 * 1 μL Leupeptin (1 mg/mL)
 * 1 μL Pepstatin (A) (1 mg/mL)
 * 5 μL Na3VO4
 * 5 μL NaF (200 mM)
 * PBS
 * Coomassie (Bradford) Protein Assay Kit
 * BCA Protein Assay Reagent (bicinchoninic acid)

Determination of protein concentration

 * Use cells put on S0 23Feb2010
 * Put cells on ice
 * Remove medium
 * Wash twice with 5 mL cold PBS
 * Add 1 mL RIPA buffer
 * Scrape of cells and collect in 1.5 mL tube
 * Sonicate (4x short pulse)
 * Take 10 μL of undiluted and a 5x diluted samples for Bradford assay (Pierce determination)

Co-immunoprecipitation

 * resuspend beads prepared 23Feb2010
 * Prepare for all donors (D9 & D12)
 * 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)
 * 5) Beads (30 μL) with only sample (200 μL))
 * Incubate ON @ 4 °C

hTERT stimulation

 * Remove medium form 24-wells plate with ASMC's grown ON in DMEM (S0)
 * Rinse twice with warm PBS and remove buffer
 * Add 200 μL DMEM S0 with either HT31 or S0 (see schedule below)
 * Incubate 20 min.
 * Add 400 μL DMEM S0 to CTR (lane A)
 * Add 200 μL DMEM S0 to CSE (lane B)
 * Add 200 μL 8-pctp or 6-benz according to schedule below (lane C, D)
 * Wait 25 min.
 * Prepare 25 mL DMEM S0 with 100% CSE
 * Dilute 100% CSE to 45% CSE
 * Add 200 μL 45% CSE to wells according to schedule below (lane B, C D)
 * Incubate 24 h.

CTR = control, CSE = Cigarette Smoke extract, 8-pcpt = 8-pcpt-2'-o-me-camp

Western blot, secondary Antibody

 * Wash 3x 10 min. in TBST buffer (first can be less)
 * Treat with secondary antibody (anti-Mouse) (diluted in 5% TBST-milk solution) (1.5h, RT)
 * Wash 3x 10 min. in TBST buffer

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 two overhead sheets
 * Take picture (depends on what is available)

Results

 * Co-IP Blots of samples pulled down using PKA antibody coated beads, blots were treated with anti-AKAP antibody

Gel 1 Gel 2
 * 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

Conclusion

 * All Co-IP samples incubated with sample (and antibody) showed a double band it is unclear wether or not it is at the right height. The used antibody was directed against AKAP179, 86 kDa

Related entries

 * Co-IP part I & Western blot

Same actions

 * Cell lysis & Co-IP Day II
 * Cell stimulation


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