User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/06/21

Summary

• 12June2010: CSE & S⁰ (hTERT D9 P27) C1 / S1
• 15June2010: CSE & S⁰ (hTERT D9 P28) C2 / S2
• 17June2010: CSE & S⁰ (hTERT D9 P29) C3 / S3
• Got some new HEK293 cells, in about 7.5 mL (2·10⁶ cells/mL) Medium
  • Spinned down 1000 rcf 5 min.
    • NOTE: First 4000 rcf for a few seconds! (maybe not nice for cells, but needed to lyse them anyway)
  • Resuspended the cells twice in cold PBS (non-sterile) followed by spinning down (1000 rcf, 5 min.) and removing supernatant
  • Added 2 mL of RIPA lysis buffer and sonicated them

Bradford assay

• Dilute samples 5x (4 μL samples + 16 μL H2O)
• Put (3x) 5 μL sample on 96-wells plate
• Put STND on plate
• Add 250 μL Bradford reagent
• Measure (AG KLUSSMANN FRANK BRADFORD ASSAY) (within 20 min.)
  • NOTE: After dilution everything was performed by Tina, Anita's "Ausbildung" student

cAMP precipitation

• All samples were diluted using MillQ water to an end concentration of 500 μg/mL
  • Dilution was prepared in a volume of 3 mL
• Each sample (1 mL) was put to 30 μL of 8-AHA-cAMP agarose beads in triplicate
• Samples were incubated with beads ON @ 4 °C

Notes

• Samples seem to have a slight precipitate, probably due to the sonication

Results

• See attachment for Bradford results

Discussion

• RIPA control for Bradford showed a high value, but seeing as this was only used in the new HEK293 preparation (and this concentration is also higher than the others) it was not taken in account for correction. Only the old HEK293 lysate was used for cAMP precipitation.

Attachment

Bradford results