The Thermo Scientific Pierce Far-Western Blot Kit for Biotinylated Proteins enables in-gel or on-membrane Western blot detection of target proteins via interaction with any biotin-labeled test protein used as the primary probe.
This far-Western blotting kit provides the reagents and protocol necessary to probe for target proteins by virtue of their interaction with a specific binding partner, which the researcher supplies in purified, biotinylated form. The kit is successful for analysis of protein-protein interactions in which the target (prey) proteins can be bound after they have been separated by gel electrophoresis. The kit includes reagents and procedures for probing and chemiluminescent detection with blots or directly from mini gels.
- On-membrane or in-gel detection options – on-membrane detection offers greater sensitivity; in-gel detection is faster and prevents problems associated with incomplete or inefficient transfer
- Nonradioactive alternative for far-Western analysis – reliable and sensitive biotin/streptavidin-HRP chemistry combined with chemiluminescent detection offers a practical and safe alternative to radiolabeling the bait protein
- Useful interaction range – effective for interaction pairs that have moderate to strong binding associations between the prey and the biotinylated bait protein
- Primary antibody-free detection – uses a biotinylated protein as the probe, eliminating the need for primary antibodies or even to have prior knowledge of target-protein identities
- Compatible with both SDS-PAGE and native gels – provides option to probe for prey proteins in a more native environment because reduced or denaturing systems may not always allow for the intended interaction
- Reduced nonspecific binding – biotin/streptavidin-HRP systems demonstrate less nonspecific binding compared to antibodies directed against the bait protein
- Compatible with protein staining – gels can be used for total protein staining after the in-gel chemiluminescent detection step, eliminating the need to run two gels
- Streptavidin-HRP conjugate, dilution and wash buffers, chemiluminescent substrate solutions, and cellophane sheets for mini gels
- Protein interaction discovery – search and screen for proteins in cell lysates that have binding affinity to the purified biotinylated protein
- Protein interaction confirmation – characterize and identify the presence of native or off-target binding interactions involving a given biotinylated protein and particular samples
Far-Western blotting involves probing for targets first with a putative interaction-protein rather than a primary antibody. The method is called far-Western blotting because primary antibodies are used secondarily to detect the primary interaction probe rather than the target protein. The Pierce Far-Western Blot Kit for Biotinylated Proteins eliminates the use of antibodies altogether. The primary probe (i.e., the biotinylated bait protein supplied by the researcher) is secondarily detected with streptavidin-HRP conjugate, whose activity is visualized by reaction with a very sensitive chemiluminescent substrate.
|Protocol scheme for the Thermo Scientific Pierce Far-Western Blot Kit for Biotinylated Proteins. The method allows identification and characterization of protein interactions, whether native or artifactual.
- Reddy, V.M. and Kumar, B. (2000). Interactions of Mycobacterium avium complex with human respiratory epithelial cells. J.I.D 181,1189-1193.
Protein Methods Library – Overview of Protein-Protein Interaction Analysis
Protein Methods Library – Far-Western Blot Analysis