Western Blot Analysis by SDS

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