Prestige Antibody Western Blot Procedure
Some Prestige Antibodies® are verified as primary reagents for Western blot analysis using the described procedure.
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
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.
- Protein samples are loaded onto Criterion TGX Precast Gels, 4–20% polyacrylamide. The electrophoresis is run according to manufacturer’s protocols.
- 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.
All incubation and wash steps are performed at room temperature and with agitation.
- 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.
- 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.
- To remove residual primary antibody, the membranes are washed 3 x 5 minutes in TBST (TBS with 0.1% (v/v) TWEEN 20).
- 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.
- To remove residual secondary antibody, the membranes are washed 4 x 5 minutes in TBST.
- The membranes are incubated with detection reagent for 1 minute.
- The image is captured using a CCD camera.
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.