User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/08/13

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SDS-PAGE Lysates, strip blot Main project page
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Summary

Materials & Methods

SDS-PAGE

  • Lysate samples were prepared, undiluted samples (75 µL) with NuPage loading buffer + DTT (25 µL)
  • Samples were put to 70 °C for 10 min.
  • Samples were put to 4-12% Gradient NuPage gel 200 V
4-12% Gradient NuPage Gel loading LYSATES hTERT D9 + HEK293, HeLa -S & Jurkat see codes 21June2010
# 1 2 3 4 5 6 7 8 9 10 11 12
Gel
Lysates
X Invitrogen Marker
(8 μL)
Jurkat
(20 μL)
HeLa -S
(20 μL)
HEK293
(20 μL)
Biorad Marker
(8 μL)
S1
(20 μL)
S2
(20 μL)
S3
(20 μL)
C1
(20 μL)
C2
(20 μL)
C3
(20 μL)

Stripping blot

  • Stripping solution
    • 50 mL dH2O
    • 5 mL Tris-Cl (1.0 M, pH 6.8) (Stacking gel buffer SDS-PAGE)
    • 1 g SDS
    • 150 mg DTT (Add later)
  1. Prewarm buffer (w/o DTT) for 30 min. to 70 °C in waterbath
  2. Add DTT just before adding membrane
  3. Completely immerse membrane
  4. Incubate 30 min @ 70 °C
  5. Wash 3x with dH2O or TBS-T
  6. Membrane can now be blocked again

Results

  • Gel after blotting


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