Far-Western Blot Analysis Service

Far-western blot is a molecular biology method based on western blot technology that can be used to detect protein-protein interactions in vitro, especially interactions that do not require the natural structure of the target protein.

 

The overall workflow of the far-western blot technique is similar to that of western blot, except that western blot detection of target proteins requires antibodies, whereas far-western blot analysis does not require antibodies but uses purified and labeled "bait" proteins to detect and identify proteins on the membrane. In both western blot and far-western blot, proteins are separated using SDS-PAGE and native PAGE gels, and then transferred from the gel to a membrane. After transfer, the membrane is blocked with BSA. The proteins on the membrane are denatured and renatured, followed by subsequent detection steps. The far-western blot technique uses commercially available highly purified bait proteins to label the membrane with probes. After the bait protein reacts with the target protein, bands corresponding to the target protein can be detected depending on the bait protein used.

 

To identify proteins that interact with a given protein, far-western blot is an effective platform with low experimental costs and high sensitivity. The principle of far-western blot is an improvement on western blot, using naturally folded recombinant proteins as the first probe, which interact with proteins transferred to a PVDF membrane. The second probe used in far-western blot is a specific antibody against the given protein, and the third enzyme-labeled probe is against the second probe. When combined with LC-MS/MS protein identification, the obtained protein spots (or bands) can be characterized, followed by further functional analysis, such as protein immunoprecipitation.

 

When analyzing far-western blot, attention should be paid to the following issues when the natural conformation and natural interactions of proteins need to be preserved: 1) Proteins separated under denaturing conditions in SDS-PAGE may denature, and denatured proteins may not react with the bait protein, leading to a failure to recognize interactions. 2) Denaturing and renaturing proteins before binding may also cause conformational changes in the protein, affecting the binding of the target protein to the bait protein. 3) If the binding site is affected by other factors and loses the ability to bind to the bait protein, the bait protein may not react with it, which can also lead to a failure to detect interactions. More troublesome is that proteins presented in a non-natural conformation may interact in new non-natural ways, leading to false positive detections.

 

MtoZ Biolabs provides far-western blot analysis services, which can be customized according to your needs and assess potential factors that may affect the results. We promise that the data provided is reliable and highly reproducible.

 

Service Advantages

1. Suitability for In Vitro Protein-Protein Interaction Studies

2. Analysis of Protein Binding Domains

3. Comparison of Protein Binding Affinity

4. No Specific Antibodies Required

 

Analysis Workflow

1. Quantification and Separation of Proteins through SDS or Native PAGE

2. Transfer of Proteins from the Gel to the Membrane

3. Denaturation and Renaturation of Proteins to Ensure Full Exposure of Binding Sites on the Membrane

4. Blocking of the Membrane with BSA Buffer

5. Incubation of the Membrane with Purified Bait Proteins

6. Detection of Bait Protein Signals

7. Delivery of Image Analysis and Report