The image is captured using a CCD camera.The membranes are incubated with detection reagent (Immobilon Western Chemiluminescent HRP Substrate, Millipore Corporation, Billerica, MA, USA) for 1 min.To remove residual secondary antibody, the membranes are washed 4 x 5 min in TBST.The secondary antibody (for monoclonal antibodies: HRP-conjugated Goat Anti-Mouse Immunoglobulin for polyclonal antibodies: HRP-conjugated Swine Anti-Rabbit Immunoglobulin, Dako, Glostrup, Denmark) is diluted 1:3000 in blocking buffer and incubated with the membranes for 30 min.To remove residual primary antibody, the membranes are washed 3 x 5 min in TBST (TBS with 0.1% (v/v) Tween20).Optimal dilution must be determined by the user. NOTE: The recommended working dilution of the primary antibody is to be considered as a guideline only. The primary antibody is diluted in blocking buffer and incubated with the blocked membranes for 1 h.To prevent non-specific background binding of the primary and/or secondary antibodies to the membrane, membranes are blocked in a bovine serum albumin-based blocking buffer (2% (w/v) in TBS with 0.1% (v/v) Tween20) for 45 min. Dried membranes from previous steps are activated in methanol for 20 seconds.The proteins are transferred from the gels to PVDF membranes through semi-dry transfer using Trans-Blot® Turbo transfer system (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol.Īll incubation and wash steps are performed at room temperature and with agitation.The electrophoresis is run according to the manufacturer’s protocols. Protein samples are loaded onto Criterion TGX Precast Gels, 4–20% polyacrylamide (Bio-Rad, Hercules, CA, USA). ![]() 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 Tris-HCl), supplemented with DTT to a final concentration of 50 mM, and incubated in 95☌ for 5 min. Electrophoresis and blotting Sample preparation Note: This protocol is the recommended Western blot protocol for the Anti-FOS antibody (AMAb91417) as well as the Anti-KLF4 antibodies (AMAb91388 and AMAb91389). Semi-dry transfers allow fast, efficient, economical blotting without a buffer tank. ![]() The Trans-Blot SD Semi-Dry Electrophoretic Blot Cell includes the cell, a Trans-Blot SD agarose gel support frame, and four packs of extra-thick blotting paper (60 sheets each, 7 x 8.4 cm, 8 x 13.5 cm and 30 sheets each, 14 x 16 cm, and 18 x 18.5 cm).Download a pdf version of the protocol for Western Blot - BSA Blocking. experience, Bio-Rad is considered the industry leader in providing. ![]() The safety cover cuts off electric current when lifted, preventing electric shock. The additional plastic templates required by other semi-dry blotters to prevent electrode shorts are unnecessary. Platinum coated titanium anodic plate electrodes and stainless steel cathode plate electrodes provide smooth and reliable transfers, durability and long life. The one-step locking system allows for quick and easy installation. Minimal buffer requirements save money Ability to transfer up to four precast or hand-poured Mini-PROTEAN® gels, or three Criterion™ gels. Features and Benefits Transfers are completed in just 15-60 minutes. ![]() The Trans-Blot Semi-Dry (SD) Transfer Cell provides fast, efficient, and cost-effective transfer without buffer reservoirs or gel cassettes.
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