User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/25

Summary

The samples have been incubated with the (PKA) antibody coated beads and today the beads are washed and stored in Laemli buffer

Materials & Methods

Materials

• Laemli buffer
• PBS
• syringe
• Hot lysis buffer

Method

Co-immunoprecipitation

• Centrifuge sample incubated with beads, 5 min. 14.000 RPM
• Remove supernatant^* (w. syringe)
• Add 1 mL PBS
• Centrifuge, 5 min. 14.000 RPM
• Remove supernatant^*
• Add 75 μL Laemli buffer to beads
• Store samples @ -20 °C
  • Alternatively go directly on to Western blot (Boil samples and spin beads down, bring supernatant onto gel)

^* Be careful not to touch beads

VASP

• Use cells grown by Bachelor students TODO
• Remove medium
• Wash with PBS twice and remove
• Add 600 μL S⁰ or S⁰+Ht31 (see hTERT VASP schedule below)
• Incubate 15-20 min. RT
• Add 600 μL S⁰ or S⁰+6-Bnz (see hTERT VASP schedule below)
• Incubate 10 min. RT
• Add 600 μL S⁰ or S⁰ 45% CSE (see hTERT VASP schedule below)
• Incubate 10 min. RT
• Remove medium
• Wash with PBS and remove
• Add 200 μL Hot Lysis buffer and scrape cells with tip
• Transfer cell lysate into 1.5 mL tubes
• Store @ -20 °C

Coating of plate

• Prepare coating buffer
  • 1.24 g Na2CO3 in 100 mL (Buffer A)
  • 2.52 g NaHCO3 in 300 mL (Buffer B)
  • Take 70 mL of Buffer A
  • Add Buffer B until pH of 9.6 has reached (150 - 200 mL)
• Continue as described 18Feb2010

BSc.

• Alamar blue
• Stop stimulation (hTERT)
• Cells to 96-wells (hTERT)

Results

• VASP blot, Anouk
• Viability staining

Related entries

Run 2: Ht31 Dose/Effect Curve & S⁰ D9/D12

• Counting cells 19Feb2010
• Putting to S⁰ 23Feb2010
• Stimulating cells 24Feb2010

Same action

• ELISA

Related topics

• SDS_sample_buffer
• Sauer:bis-Tris_SDS-PAGE,_the_very_best
• Odom:Western_Blot
• Carrico:Recipes
• Griffitts:Common_buffers
• Griffin:Antibody_Related_Solutions_&_Recipes