Ketner: Tissue culture viral lysate Western Blot

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(Materials)
(Procedure)
Line 15: Line 15:
**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 12:30, 6 September 2006

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

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
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