Western Blot Analysis by SDS

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Materials
Ready Gel Precast Gels
Bio-Rad Mini-Protean-3 Cell apparatus
Prestained protein markers

Reagents
6x Laemmli reducing sample buffer
Distilled H20 (dH20)
Running buffer
Cold Transfer buffer

Before you start:
Turn on heat block to 95-100°C
Make sure ice block cooling unit is prepared for transfer

Procedure
1. Prepare Ready Gel Cassette:
Remove the Ready Gel from storage pouch
Gently remove the comb and rinse the wells thoroughly with distilled water or running buffer
Cut along the dotted line at the bottom of the Ready Gel cassette with a razor blade
Pull the clear tape at the bottom of the Ready Gel Cassette to expose the bottom edge of the gel
2. Assemble Electrophoresis Module (see Mini-Protean 3 cell assembly guide)
3. Sample preparation:
a.Total volume to be loaded: 30ul
x ml of sample (use 30ug total protein/well)
5 ml 6x Leammlis sample buffer
y ml dH20 (for a total volume of 30ul)
b. Heat samples @ 95°C for 5 min. briefly spin
c. Load protein marker to end well
d. Load 30µl sample with loading tiPS
Note: carefully load lanes so samples don’t spill into adjacent lanes
4. Gel Electrophoresis
Mini Tank Assembly
Put Green lid on (red/red and black/black)
- Power Supply
oTurn ON
o Constant Volt
- Run @ 120V ~10min (get samples through stacking gel)
- Run @ 100V until marker is near bottom of gel (~1.5 hrs)

Transfer
Additional Materials
Glass Pyrex Dish
Clear/Black sandwich Press
Sponges
Whatmann filter paper
Forceps to handle membrane
Nylon Membrane
NO HANDS ON MEMBRANE

Cut filter paper and membranes to size
Ice block (located in–20°C freezer)
Small stirring bar