Bitan:wb

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This is a method for detecting Methionine Sulfoxide (MetO) in human plasma samples via Western Blot.

Contents

Ingredients

Buffers

Tris-Glycine Sample Buffer

63 mM Tris HCl 10% Glycerol 2% SDS 0.0025% Bromophenol Blue pH 6.8

Tris-Glycine Running Buffer

25mM Tris base
192mM Glycine
pH 8.3

Tris-Glycine Transfer Buffer

12 mM Tris Base
96 mM Glycine
pH 8.3
Mix four parts Transfer Buffer with one part Methanol before transfer


PBST
Blocking Buffer

Reagents

  • Amersham ECL Advance working reagent
  • Human plasma (total protein 25ug/ul)
  • Novex 10 well 4-20% tris-glycine gels
  • Nitrocellulose membranes 0.22uM pore size

Recipe

Electrophoresis

  • Prepare 1L Tris-Glycine Running Buffer
  • Open pre-cast gels - remove comb and tape
  • Rinse unpolymerized acrylamide from the gel's wells with Running Buffer
  • Assemble the gel box - seal the box and test the seal with some Running Buffer
  • Denature MW ladder (Benchmark protein standard) at 95oC
  • Prepare plasma samples to 25ug/ul total protein and dilute 1:1 in Sample Buffer
  • Load 4ul protein sample per lane
  • Load 1ul protein standard per lane
  • Any empty lanes should be loaded with 4ul Sample Buffer
  • Fill the gel box's middle chambers and electrophorese at 100V for 2-3h

Transfer

Prepare 1L Tris-Glycine Transfer Buffer
Make nitrocellulose "sandwiches"

  • fiber pad
  • blotting paper
  • nitrocellulose membrane
  • polyacrylamide gel
  • blotting paper
  • fiber pad

proteins will migrate from black to red so sandwich accordingly
The transfer gel box needs ice and a stir bar to keep the temperature down
transfer should be done in the cold room (200V and 0.4A for 40min)

Blocking

Prepare 10ml Blocking Buffer per gel
Use clean tweezers to put the membrane in a clean plastic dish and add Blocking Buffer
Incubate 2h at room temperature with shaking

Primary Antibody

Decant blocking buffer from the plastic dish(es)
Dilute Anti-MetO antibody 1:1000 in Blocking Buffer
Add >=5ml antibody solution to each
Incubate 60-90min at room temperature with shaking
Decant antibody solution

Secondary Antibody

Perform four 10min washes with PBST
Decant wash buffer
Dilute anti-rabbit-HRP secondary antibody 1:10000 in Blocking Buffer
Add >=5ml antibody solution to each dish
Incubate 60-75min at room temperature with shaking

ECL Development

Perform four 10min washes with PBST
Decant wash buffer
Mix equal parts Solution A and Solution B from Amersham ECL Advance kit (need 400ul per membrane)
Add 400ul ECL working reagent to a membrane and develop with film or in an AlphaImager

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