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Western Transfer

Western transfer, or Western Blotting, involves protein transfer from polyacrylamide gels to a PVDF or nitrocellulose membrane to allow detection by specific antibodies. G-Biosciences offers high quality reagents and resources for the entire Western transfer process to ensure successful transfer and clean protein detection.

Protein Transfer

For the initial transfer, G-Biosciences has high protein binding membranes (PVDF and nitrocellulose) and high efficiency Western Transfer Buffers, including a buffer specifically designed for high molecular weight protein transfer. Following protein transfer, it’s highly recommend to check protein transfer efficiency with a rapid and reversible protein stain, such as our Swift™ Membrane Stain or BLOT-FastStain™. For improved protein transfer or for difficult to transfer proteins, such as high molecular weight proteins, G-Biosciences has Western Transfer Pads for improved transfer efficiency.

Western Blotting

For probing the Western Transfer Membranes, the first step is to block non-specific binding sites with an appropriate blocking agent. G-Biosciences offers a whole panel of blocking agents, including non animal protein blocking agents (NAP-BLOCKER™ or Protein-Free™ blocking agent), non-sera animal protein blocking agents (Superior™ Blocking Agent or FISH-Blocker™, and standard blocking solutions using milk, casein or BSA. A convenient Blocking Agents Trial Pack is offered to determine the best blocking agent to use.

Once the membrane is blocked, a primary antibody of choice is added. G-Biosciences offers over 21,000 primary antibodies that can all be directly conjugated to HRP or other detection probes. Alternatively, a secondary antibody conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) can be used to detect the primary antibody. Following incubation of the primary and secondary antibodies, the membranes need to be extensively washed with wash buffers (TBS-Tween or PBS-Tween).

Protein Detection

The final step is protein detection, where the detection probe is visualized via chemiluminescence or chromogenic reactions. Our femtoLUCENT™ PLUS and picoLUCENT™ PLUS are highly sensitive chemiluminescence reagents for HRP and AP, whereas femtoCHROMO™-AP and femtoCHROMO™-HRP allow for chromogenic detection of proteins.

Our Western ReProbe™ products allow you to strip your blot and reprobe with different antibodies, such as housekeeping antibodies, and our and Swift™ Film Cleaner helps clean spotty and overexposed films.

Visit our partner, BT Lab Systems, for Protein Electrophoresis and Western Blotting equipment, including semi dry transfer units.

 

Download the Western Blotting Handbook for Troubleshooting Tips
Protocols.io provides an interactive version of this protocol where you can discover and share optimizations with the research community.    picoLUCENT™ PLUS-HRP kit is based on our ultra sensitive Luminol substrate that produces chemiluminescence upon reaction with horseradish per..
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Protein-Free Blocking Buffer does not contain protein; it is a proprietary formation of non-protein agents that eliminates non-specific binding sites in ELISA, blotting, immunohistochemistry and other applications.  The absence of protein from the Protein-Free Blocking Buffer eliminates problem..
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 Our PVDF (polyvinylidene) membranes are pre-cut membranes for Western transfers, or Western blotting.  The PVDF membranes bind biomolecules, including proteins, through hydrophobic interactions The presence of the 0.45µm pore sizes increases the surface binding area. Typical thickn..
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We offer a range of secondary antibodies conjugated to either alkaline phosphatase (AP) or horseradish peroxidase (HRP). The antibodies are isolated from antisera by immuno-affinity chromatography using antigen coupled to sepharose beads or other methods. They are supplied lyophilized, from a buffer..
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Superior™ Blocking Buffer contains a proprietary antiginically non-determinant protein for blocking non-specific sites during ELISA, membrane blotting, immunohistochemistry and other applications.    Superior™ Blocking Buffer is ideal for a high signal to background ratio in ..
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The Swift™ Western Blotting System is a unique system that reduces the blocking and antibody incubations on Western blot membranes from >4 hours to <60 minutes.    Using a combination of proprietary wash and diluent buffers and our highly sensitive femtoLUCENT™ chemilum..
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The Swift™ Western Diluent is a unique solution that reduces the blocking and antibody incubations on Western blot membranes from >4 hours to <60 minutes.     Swift™ Western Diluent generates comparable result to traditional Western blotting procedures and other commerc..
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 A 10X Concentrated solution of Tris Buffered Saline with Tween® 20.    The 10X Concentration is 100mM Tris.HCl, 150mM NaCl, 0.5% Tween® 20 at pH7.5.   The 1X Concentration is 10mM Tris.HCl, 15mM NaCl, 0.05% Tween® 20 at pH7.5.   TBST is commonly ..
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Western ReProbe™ is a single component system that is specifically formulated to dissociate and remove antibodies from membrane-bound proteins without destroying the antigenic binding affinity. Western ReProbe™ also enables the ability to reuse western blots. Western ReProbe™ brea..
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Based on our popular Western ReProbe™, the modified formulation allows for the removal of stubborn, high affinity antibodies from membrane bound proteins without destroying the antigenic binding affinity.  The membrane bound protein is retained on the membrane and the matching antibodies ..
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The Blocking Agents Trial Packs are a set of four G-Biosciences proprietary blocking agents for immunoassays with one being a non-animal protein blocker and another protein free that minimizes the risk of non-specific binding of antibodies during the immunoassay. Each blocking buffer provided in the..
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BLOT-QuickBlocker™ is a novel modified milk protein that is highly soluble and does not inhibit peroxidase detection. The modified milk protein has high blocking efficiency with a clear background. Features   Readily soluble and produces semi-clear solution. No inhibition to peroxi..
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