Bitan:wb

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

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
=Recipe=
 * 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

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" 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)
 * fiber pad
 * blotting paper
 * nitrocellulose membrane
 * polyacrylamide gel
 * blotting paper
 * fiber pad

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