Far-Western Blot Service

Most proteins in living cells are thought to function by interacting with other proteins or in complexes. Therefore, the study of protein-protein interactions is essential to understand the function of proteins within the cell. In the past decades, several methods have been developed to detect protein-protein interactions. Among the existing methods for detecting protein-protein interactions, far-western blot has been widely used for the study of protein-protein interactions in prokaryotes and eukaryotes.

Far-western blot is similar with western blot in which membranes are probed directly with specific antibodies, and is also referred to as a west-western or blot overlay assay. In contrast to western blotting where a target protein is usually known in advance, far-western blotting can detect proteins on the basis of presence or absence of binding sites without any previous knowledge about their identities.

Far-Western Blot for Protein–protein Interactions Detection

Far-western blotting is now used to study protein-protein interactions, such as receptor-ligand interactions, and to screen libraries for interaction partners.

Far-western blotting is widely used to (i) analysis of interactions between known proteins. (ii) analysis of interactions between known and unknown proteins. Since cell extracts are usually completely denatured by boiling in detergent prior to gel electrophoresis, this method is best suited for detecting interactions that do not require the natural folded structure of the desired protein.

Our Far-Western Blot Service

Far-western blot is an effective platform with low cost and high sensitivity. And when Far-western blot is combined with LC-MS-MS protein identification, the resulting protein spots (or protein bands) can be characterized and then subjected to further functional analysis, such as protein immunoprecipitation.

Depending on your needs, Creative Proteomics offers custom Far-western blot analysis services for detecting protein interactions. Also in combination with the LC MS platform, we can provide further functional analysis with reliable and reproducible data.

Advantages of Far-Western Blot Analysis

• Can be used for in vitro protein interaction studies
• Analyze protein binding domains
• Compare protein binding affinity
• No specific antibody required
• good repeatability;
• Multiple tissue samples can be analyzed at once;

Disadvantages of Far-Western Blot Analysis

• Experimental procedure involves multiple washing steps and weak interactions are difficult to detect.
• Interactions are difficult to detect if the target protein is low in the tissue.
• Experiments may involve protein denaturation and renaturation, and natural structure-dependent interactions cannot be detected.

Workflow for Far-Western Blot Analysis Service

1. Protein quantification and separation by SDS or native PAGE

2. Transfer the protein from the gel to the membrane

The purpose of this step is to attach the protein to the surface of the membrane so that the protein becomes easily detectable. It is important to avoid contamination during the membrane transfer process, as the final assay will have a high background if contamination is present during the transfer process.

3. Denature and renature the protein to ensure adequate exposure of protein binding sites on the membrane.

4. Block the membrane with BSA buffer.

It should be noted that the blocking agent may cross-react or disrupt the interaction of the protein to be studied in other ways, so a suitable blocking agent needs to be determined empirically.

5. Incubate the membrane with purified bait protein

Probe proteins can usually be produced using E. coli expression systems, and although cell lysates can also be used as probe proteins, it is best to choose purified proteins as probes in order to reduce the experimental background (the higher the purity of the bait protein, the higher the success rate of the experiment).

6. Detection of bait protein signal

The following strategies are commonly used to detect probe:

Unlabeled bait protein → enzyme-labeled bait-specific antibody → substrate reagent

Radiolabeled bait protein → exposure to film

Biotinylated bait protein → enzyme-labeled streptavidin → substrate reagent

Fusion-labeled bait protein → tag-specific antibody → enzyme-labeled secondary antibody → substrate reagent

7. Experimental control settings

In order to improve the accuracy of the results, it is necessary to set appropriate experimental controls. For example, if the bait protein is a GST-fusion protein, a separate group of GST-tagged experimental groups is set as a negative control to exclude the possibility of non-specific binding of the GST tag itself to the target protein on the membrane.

8. Data analysis and report delivery