Ketner: Tissue culture viral lysate Western Blot
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Current revision (14:03, 6 September 2006) (view source) (→Materials) |
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*[[PBS-T]] | *[[PBS-T]] | ||
*5% milk in PBS-T | *5% milk in PBS-T | ||
| + | *30% Acrylamide | ||
| + | *1.5M Tris pH 8.8 | ||
| + | *10% SDS | ||
| + | *10% APS (fresh) | ||
| + | *TEMED | ||
| + | |||
| + | |||
| + | |||
| + | ---- | ||
===Procedure=== | ===Procedure=== | ||
| Line 15: | Line 24: | ||
**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]] | ||
| + | *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 | ||
Current revision
Contents |
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
