Far-Western Blot Analysis Service Online Inquiry

Introduction

Far-WesternBlot is a molecular biological  method, which is based on the technique of Westernblot, to detect in vitro  protein-protein interactions, especially for detecting interactions that do not  require the native structure of the protein of interest.

The overall workflow of far-Western blot  technique is similar to Western blotting, except that antibodies are needed for  probing target proteins in Western blotting. Whereas in far-Western analysis, antibodies  are not needed, instead a purified and labeled "bait" proteins are  used to probe and detect a target "prey" protein on the membrane. In  both Western blotting and far-Western blot, proteins are separated either SDS  gel of native PAGE gel, followed by transferring proteins from the gel onto the  membrane. After transfer, the membrane is blocked with BSA with empirical  concentration. Then proteins on the membrane are denatured and renatured, which  is an optional step and depends on the binding conditions needed for the  experiments. The membrane is then probed with a known highly purified bait  protein. Following the reaction of the bait with the prey protein, a detection  system, dependent upon the bait protein used, signals of band that corresponds to  the prey protein can be detected.

Critical  factors of Far-Western Blot Analysis

Though Far-Western blot is a convenient way  and has been widely used to study protein-protein interaction, cautions must be  taken when preserving the native conformation and interaction conditions for  the proteins. There are a few problems regarding far-Western blot. First, for  proteins separated in a SDS gel, which is a denaturing condition, proteins are  denatured. It is possible that denatured proteins may not interact with bait  proteins, resulting in a failure to identify an interaction. Secondly, proteins  are denatured and renature before binding assay, which may also leads to a  potential conformational change of the protein, affecting the binding affinity  of target proteins to the bait protein. Thirdly, if the exposure of binding  motif is regulated by other factors, there can be a chance that the binding  motif is buried inside and become unreachable to the bait, and cause a failure  of detection of interaction. What’s more tricky is that, proteins presented in  non-native conformations may interact in novel, artificial ways, resulting in  false-positive interactions. Creative Proteomics can design the experiment  based on your needs, and assess the potential factors that may affect results.  We promise reliable data with high reproducibility.

Di-Sulfide Bond Localization

Features  of Far-Western Blot Analysis

  • Study in vitro protein  interactions
  • Map protein binding domain
  • Compare protein binding  affinity
  • No requirement for antibody

Workflow  of Far-Western Blot Analysis

  • Protein are quantified and  separated by SDS or native PAGE
  • Transfer proteins from gel to a  membrane
  • Transferred proteins are  denatured and renatured to fully expose the binding motif
  • The membrane is then blocked  with BSA buffer
  • The membrane is incubated with  purified bait protein
  • Signals of bait proteins are  detected
  • Images analysis & report  delivery

Our Far-Western Blot methods contain:

  • Direct detection of prey  protein with a radioactive bait protein;
  • Indirect detection with an  antibody specific to the bait protein;
  • Indirect detection with an  antibody specific to the tag of a fusion-tagged bait protein;
  • Indirect detection with a  biotinylated bait protein and enzyme-conjugated avidin or streptavidin.

Ordering Procedure:

Ordering Procedure

* For Research Use Only. Not for use in diagnostic procedures.
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