User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization

ELISA

1. Viability & ELISA
2. Dose effect curve ELISA
3. ELISA
4. ELISA
5. ..
6. ELISA Berlin Okay

Co-IP

1. Co-IP 1 Anouk
2. Co-IP 2 Anouk
3. Co-IP 3 Anouk
4. Co-IP 4 TODO

RII Overlay

1. ..
2. RII overlay Okay

Misc.

• Ht31P toxicity
• Statistical analysis

February

• 10/02/2010: Splitting ASM cells
• 12/02/2010: Counting cells
• 15/02/2010: Putting cells to S⁰
• 16/02/2010: Stimulation hTERT cells
• 17/02/2010: Viability staining
• 18/02/2010: ELISA
• 19/02/2010: Counting and splitting cells
• 22/02/2010: Preparing new DMEM S⁺
• 23/02/2010: Putting cells to S⁰, Co-immunoprecipitation
• 24/02/2010: Co-immunoprecipitation part II, (Stimulation hTERT cells v2)
• 25/02/2010: Co-immunoprecipitation part III & Viability staining
• 26/02/2010: ELISA, counting/splitting cells

March

• 01/03/2010: Western blot
• 03/03/2010: Co-IP day I, GAPDH, VASP, Stop Stimulation HT4
• 04/03/2010: Co-IP day II, ELISA HT4, Cell Stimulation
• 05/03/2010: Co-IP day III, Stop stimulation hTERT, Split hTERT
• 08/03/2010: Western Blot
• 09/03/2010: hTERT stimulation @ splitting, Western blot contn'd
• 10/03/2010: Start stimulation D9, Stop stimulation D12
• 11/03/2010: Stop stimulation
• 12/03/2010: Splitting & Counting cells
• 15/03/2010: Co-IP & ELISA prep
• 16/03/2010: ELISA & hTERT stimulation
• 17/03/2010: Ht31P toxicity
• 18/03/2010: Statistical analysis
• 22/03/2010: Co-IP & Western blot
• 23/03/2010: Co-IP Part II, hTERT Stimulation & Western blot cont'd
• 24/03/2010: Co-IP Part III, Stimulation stop/Viability staining
• 25/03/2010: ELISA
• 26/03/2010: Last Groninger protocols, cell splitting

• 31/03/2010: Building smoke machine, cell splitting

April

• 01/04/2010: Creating RII overlay protocol
• 06/04/2010: Putting cells to S⁰ and splitting cells
• 07/04/2010: Preparation first Pilot Berlin
• 08/04/2010: cAMP precipitation for RII overlay & New cells
• 09/04/2010: Western blot cAMP precipitation for RII overlay
• 11/04/2010: Splitting D9
• 13/04/2010: RII overlay, splitting cells
• 14/04/2010: RII overlay & Western blot
• 15/04/2010: SDS-PAGE (MARKERS) and Immunoblot
• 16/04/2010: Split cells for ELISA and secondary antibody Immunoblot
• 18/04/2010: Refreshed medium for ELISA cells
• 19/04/2010: Put cells to S⁰ for cAMP precipitation, looked into viability staining (MTT Assay)
• 20/04/2010: Put cells to S⁰ for ELISA, stimulation for cAMP precipitation
• 21/04/2010: cAMP precipitation, stimulation for ELISA
• 22/04/2010: Stopping ELISA stimulation, preparing gels
• 23/04/2010: ELISA
• 26/04/2010: SDS-PAGE & Refreshed medium
• 27/04/2010: RII Overlay
• 28/04/2010: RII Overlay & Split cells
• 29/04/2010: SDS-PAGE& Finalizing RII overlay
• 30/04/2010: Splitting cells, Immunoblot

May

• 03/05/2010: Split cells for RII overlay
• 05/05/2010: Put cells to S⁰ for RII overlay
• 06/05/2010: Stimulation for RII overlay & Preparation of HBBS buffer for Alamar blue
• 07/05/2010: Potter-Elvehjem Cell Homogenizing
• 09/05/2010: SDS-PAGE of cAMP precipitation
• 10/05/2010: Refreshed medium
• 11/05/2010:Split cells
• 13/05/2010:Split cells for ELISA
• 16/05/2010:Put cells to S⁰ for ELISA
• 17/05/2010:Cell stimulation for ELISA
• 18/05/2010:RII Overlay & Stop stimulation ELISA
• 19/05/2010: RII overlay cAMP precipitation
• 25/05/2010: Splitting cells
• 26/05/2010: New cells, cAMP-precipitation, AKAP79 immunoblot of cAMP-precipitation
• 27/05/2010: cAMP precipitation & AKAP79 immunodetection
• 28/05/2010: Immunoblot AKAP79 (upstate)
• 31/05/2010: SDS-PAGE for AKAP79 immunoblot & RII overlay (HEK293)

June

• 01/06/2010: SDS-PAGE for immunoblot (fail)
• 02/06/2010: Preparing TRIS Urea gel
• 03/06/2010: Tris urea gel
• 07/06/2010: Urea-TRIS page, splitting cells
• 08/06/2010: Creating RNA isolation protocol
• 09/06/2010: Refreshed medium
• 10/06/2010: Put cells to S⁰, split cells
• 11/06/2010: Cell stimulation
• 12/06/2010: Stop stimulation: Protein and RNA/Protein isolation
• 13/06/2010: Putting cells to S⁰, splitting cells and continuing of protein isolation
• 14/06/2010: Cell stimulation
• 15/06/2010: Stop stimulation: Protein and RNA/Protein isolation (2)
• 16/06/2010: Stimulation of cells
• 17/06/2010: Stop stimulation: Protein and RNA/Protein isolation (3)
• 21/06/2010: New HEK293, Bradford assay, cAMP precipitation
• 22/06/2010: cAMP precipitation
• 23/06/2010: Immunoblot
• 24/06/2010: Immunoblot repeat
• 25/06/2010: Coomassie staining
• 28/06/2010: HeLa lysis, Bradford & cAMP precipitation part I
• 29/06/2010: cAMP precipitation part II & SDS-PAGE
• 30/06/2010: Lyse HeLa

July

• 01/07/2010: Bradford, qPCR AKAP12 & AKAP9, Silverstaining
• 02/07/2010: qPCR GAPDH, Silverstaining
• 03/07/2010: Silver staining attempt #3
• 05/07/2010: Bradford & cAMP precipitation
• 06/07/2010: cAMP precipitation gel
• 07/07/2010: RII overlay cAMP precipation
• 08/07/2010: RII overlay result
• 09/07/2010: Gravin Co-IP, cAMP precipation gel
• 10/07/2010: Gravin Co-IP gel
• 11/07/2010: AKAP250 (scbt) immunoblot cAMP precipitation
• 12/07/2010: AKAP250 (scbt) immunoblot AKAP250 Co-IP
• TODO 13/07/2010: AKAP250 (Eurogentech) immunoblot
• TODO 14/07/2010: AKAP250 (abcam) immunoblot
• 15/07/2010: Epac immunoblot
• TODO 16/07/2010: Epac immunoblot result
• 19/07/2010: Epac immunoblot repeat, new cell culture
• TODO 20/07/2010: Epac immunoblot repeat result
• 21/07/2010: Splitting cells
• 24/07/2010: Splitting cells for immunofluorescence
• 25/07/2010: Cell check
• 26/07/2010: Cover slips to S⁰ & Plating out for ELISA
• 27/07/2010: Stimulation cover slips, PKA-Co-IP
• 28/07/2010: Immunofluorescence for cells, Gravin & RII localization
• 29/07/2010: PKA-Co-IP gel
• 30/07/2010: Trypsin/EDTA dilution, splitting cells for ELISA
• 31/07/2010: Put cells to S⁰

August

• 01/08/2010: Stimulation for ELISA
• 02/08/2010: Put cells to S⁰, Splitting cells, stopping stimulation
• 03/08/2010: ELISA, RII, IF
• 04/08/2010: Stop stimulation, put IF to S⁰
• 05/08/2010: Start stimulation ELISA & IF, splitting cells
• 06/08/2010: Ending stimulation ELISA, Primary antibody IF
• 07/08/2010: Finish IF, Split cells
• 09/08/2010: Isolate Protein/RNA Jurkat cells, Codes IF
• 10/08/2010: ELISA, qPCR Jurkat, SDS-PAGE cAMP precipitation
• 11/08/2010: Jurkat qPCR results, Primary antibody WB, Ending stimulation ELISA
• 12/08/2010: Lyse HEK293 (isolate RNA), 2^nd AB WB, Convocal microscopy
• 13/08/2010: SDS-PAGE Lysates, strip blot
• 14/08/2010: Secondary antibody cAMP precipitation blot of Gravin en AKAP79
• 16/08/2010: SDS-PAGE lysates, Cell splitting
• 17/08/2010: ON Blot lysate samples
• 18/08/2010: BioRad workshop
• 19/08/2010: Prepare cDNA, Immunoblot visualization, 1^st AB ON Blot
• 20/08/2010: Stripping blots, qPCR AKAP8, refresh medium new cells, 2^{nd AB}
• 21/08/2010: Lysate SDS-PAGE, qPCR AKAP5, Western blot AKAP79 and Gravin
• 23/08/2010: SDS-PAGE cAMP precipitation, pre-IgG antibody
• 24/08/2010: RII Overlay, Pre-IgG 2^nd AB, CSE on HEK293, ASMC to S⁰, Split cells w. Mileh
• 25/08/2010: RII Overlay all