User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/04/09

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Western blot cAMP precipitation for RII overlay Main project page
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Summary

  • cAMP coupled beads incubated with samples ON were washed and boiled in sample buffer. Together with lysates samples were put to pre-cast gradient (4 - 20%) gel

Materials & Methods

Materials

  • 4x Sample buffer
  • Vaccuum pump
  • RIPA buffer

Method

Washing beads: cAMP precipitation

  • Spin beads incubated ON @ 4 °C 5 min. 2100xg @ 4 °C
  • Remove supernatant with vacuum pump
  • Add 500 μL RIPA buffer
  • Spin 5 min. 2100xg @ 4 °C
  • Remove supernatant with vacuum pump
  • Add 500 μL RIPA buffer
  • Spin 5 min. 2100xg @ 4 °C
  • Remove supernatant with vacuum pump
  • Add 500 μL RIPA buffer
  • Spin 5 min. 2100xg @ 4 °C
  • Remove supernatant with vacuum pump and syringe
  • Add 20 μL of 2x sample buffer
    • Dilute 4x sample buffer 1:1 with H2O
  • Spin 5 min. 2100xg @ 4 °C

Gradient SDS-PAGE

  • Mini-Protean® TGX™ precast gel of BioRad was used
    Biorad Precision Plus Protein Dual Color marker
    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)
Gel loading
# 1 2 3 4 5 6 7 8 9 10
Gel 1
Lysates 		Marker
(8 μL) 		D9 S0
(08April2010) 		D9 CSE15%
(08April2010) 		D9 CSE15%B
(08April2010) 		D12 S0
(08April2010) 		D12 CSE15%
(08April2010) 		D12 CSE15%B
(08April2010) 		X D9 S0 (23March2010) D12 S0 (23March2010)
Gel 2
cAMP precipitation 		Marker
(8 μL) 		D9 S0
(08April2010) 		D9 CSE15%
(08April2010) 		D9 CSE15%B
(08April2010) 		X D12 S0
(08April2010) 		D12 CSE15%
(08April2010) 		D12 CSE15%B
(08April2010) 		X Loading dye

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

  • For results the membrane picture was mirrored

Results

  • Ponceau S Staining to reveal proteins tranferred onto membrane


Image 090420101: Membrane with proteins transferred of SDS-PAGE lysate samples

Image 090420102: Membrane with proteins transferred of SDS-PAGE cAMP precipitated samples

Discussion

Lysates

  • The lysate samples show nice bands of all proteins present in the cells
  • D12 seems to contain more proteins than D9, also seen in those taken from Groningen although in lesser amount
  • RII overlay should tell us which are PKA interacting proteins

cAMP precipitation

  • A strong contrast to the lysate samples at least showing that most proteins seen in the lysates ar not cAMP interacting proteins and should also not been seen later on in the RII overlay assay
  • Again D12 seems to carry more proteins
  • An >250 kDa bands seems to be present in all samples
  • It appears to show some bands also between 100 and 150 kDa but more can be said after RII overlay

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