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
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
