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Prestige Antibody Western Blot Procedure

Product Description

Some Prestige Antibodies® are verified as primary reagents for Western blot analysis using the described procedure.

Preparation Instructions

Wash Buffer – 10 mM Tris-HCl (T3253) with 150 mM NaCl (S3014) and 0.05% (v/v) TWEEN® 20 (P8416), pH 7.5

Blocking Buffer - 10 mM Tris-HCl (T3253) with 150 mM NaCl (S3014), 5% non-fat dried milk, and 0.5% (v/v) TWEEN® 20 (P8416), pH 7.5

Electrophoresis and Blotting

Sample Preparation

Protein samples (selected tissue lysates, cell lysates or over-expression lysates) are mixed with Laemmli buffer (to a final loading concentration of 2% SDS, 10% glycerol, 0.002% bromophenol blue, 0.0625 M TrisHCl), supplemented with DTT to a final concentration of 50 mM, and incubated in 95°C for 5 minutes.

Procedure

  1. Protein samples are loaded onto Criterion TGX Precast Gels, 4–20% polyacrylamide. The electrophoresis is run according to manufacturer’s protocols.
  2. The proteins are transferred from the gels to PVDF membranes through semi-dry transfer using Trans-Blot® Turbo transfer system (Bio-Rad) according to manufacturer’s protocol.

Immunodetection

All incubation and wash steps are performed at room temperature and with agitation.

Procedure

  1. Dried membranes from previous steps are activated in methanol for 20 seconds. To prevent non-specific background binding of the primary and/or secondary antibodies to the membrane, membranes are blocked in milk-based blocking buffer (5% (w/v) non-fat dried milk in TBS with 0.1% (v/v) TWEEN 20) for 45 minutes.
  2. The primary antibody is diluted in blocking buffer and incubated with the blocked membranes for 1 hour.

    NOTE: The recommended working dilution of the primary antibody is to be considered as a guideline only. Optimal dilution must be determined by the user.

  3. To remove residual primary antibody, the membranes are washed 3 x 5 minutes in TBST (TBS with 0.1% (v/v) TWEEN 20).
  4. The secondary antibody (for monoclonal antibodies: HRP-conjugated Goat Anti-Mouse Immunoglobulin; for polyclonal antibodies: HRP-conjugated Swine Anti-Rabbit Immunoglobulin) is diluted 1:3000 in blocking buffer and incubated with the membranes for 30 minutes.
  5. To remove residual secondary antibody, the membranes are washed 4 x 5 minutes in TBST.
  6. The membranes are incubated with detection reagent for 1 minute.
  7. The image is captured using a CCD camera.
Typical Western blot

Figure 1. Typical Western blot


Prestige Antibodies is a registered trademark of Sigma- Aldrich
® Biotechnology LP and Sigma-Aldrich Co. LLC
TWEEN is a registered trademark of Croda International PLC.

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