Ketner: Tissue culture viral lysate Western Blot

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(Prepare Gel)
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===
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*Boil agar to create plug
*Boil agar to create plug
*Drip agar down side of cassette with pasteur pipette, just enough to form seal
*Drip agar down side of cassette with pasteur pipette, just enough to form seal
-
*[[Create running]]
+
*[[Create running gel]]
*Layer isopropanol on top to remove bubbles
*Layer isopropanol on top to remove bubbles
*[[When hardened create stacking gel]]
*[[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

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