Ketner: Tissue culture viral lysate Western Blot: Difference between revisions
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**High mol weight 30KDa - 220 KDa | **High mol weight 30KDa - 220 KDa | ||
**Low mol weight 6.5KDa - 45 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]] |
Revision as of 10:30, 6 September 2006
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
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