Ketner: Tissue culture viral lysate Western Blot

Overview
A Western Blot allows for the semiquantitative determination of protein expression. Crude cell lysates are loaded into a polyacrylamide gel containing a denaturing agent which give all the proteins a net negative charge. A current can then be passed through the gel and the proteins will migrate through the gel, with the largest proteins traveling the slowest, resulting in a lane where proteins become separated on the basis of their weight. The proteins can then be transferred from the gel onto a membrane (often nitrocellulose or PVDF), which can then by incubated with an antibody directed against the protein of interest. By using a detector conjugated to an antibody one can now detect specifically the protein of interest.

Materials

 * 2x PLS
 * PBS-T
 * 5% milk in PBS-T
 * 30% Acrylamide
 * 1.5M Tris pH 8.8
 * 10% SDS
 * 10% APS (fresh)
 * TEMED

Prepare Samples

 * Boil Samples for 5 min, cool on ice 1 min, spin 1 min
 * Ladders: 8ul ladder + 8ul 2x PLS
 * High mol weight 30KDa - 220 KDa
 * Low mol weight 6.5KDa - 45 Kda

Prepare Gel
This will vary according to gel apparatus
 * Clean and assemble gel apparatus
 * Boil agar to create plug
 * Drip agar down side of cassette with pasteur pipette, just enough to form seal
 * Create running gel
 * Layer isopropanol on top to remove bubbles
 * When hardened create stacking gel
 * Cast stacking gel with comb, remove comb when hardened
 * Fill with 1x running buffer
 * Load samples and run at 50-120V

Transfer to membrane
specific for apparatus
 * overnight, constant voltage of 40mA

Probe Blot with antibodies

 * Block with 5% milk in PBS-T for 1hr
 * Primary in 5% milk in PBS-T for 2 hrs
 * Wash 3x with 5% milk in PBS-T for 5 min each
 * Secondary in 3% milk in PBS-T for 45 min
 * Wash 3x with 5% milk in PBS-T for 5 min each
 * Wash 1x with PBS-T for 5 min

Develop Blot and document
specific to secondary antibody