Ketner: Tissue culture viral lysate Western Blot

SDS-PAGE Westerns

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

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Procedure

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