How to Detect the Protein Ubiquitination Level via Western Blot?

Western blot can accurately detect the presence and expression of specific proteins in samples, and it can also be used to detect protein post-translational modifications, such as phosphorylation, ubiquitination, etc. The overall workflow of Far-Western Blot technology is similar to Western Blot, but the detection of target proteins in Western Blot requires antibodies, while antibodies are not needed in Far-Western Blot analysis. Instead, purified and labeled "bait" proteins are used to detect and assay the target proteins on the membrane. In Western Blot and Far-Western Blot, proteins are separated from the samples by SDS-gel and native PAGE gel, and then the proteins are transferred from the gel to the membrane. After the transfer, BSA is used for closure. The proteins on the membrane are denatured and refolded, and the subsequent detection steps are carried out. Far-Western Blot technology uses commercially highly purified bait protein to mark the probe on the membrane. After the bait protein reacts with the target protein, a band corresponding to the target protein can be detected depending on the bait protein used.

 

Experimental Materials

1. Samples: Cell or Tissue Extracts

2. Western Blot Kit with Ubiquitination-Specific Antibody and Target Protein-Specific Antibody

3. Running Gel and Transfer Membrane

4. Other Routine Western Blot Related Reagents and Equipment

 

Experimental Method

1. Sample Preparation 

Proteins are extracted with a lysis buffer containing ubiquitin enzyme inhibitors to prevent protein deubiquitination. Then, the protein samples are boiled with SDS-PAGE sample buffer to break non-covalent protein complexes.

 

2. Western Blot Analysis 

Protein samples are separated by SDS-PAGE and then transferred to a membrane. After blocking non-specific binding sites, the membrane is incubated with ubiquitin antibody and target protein antibody. After washing, incubate with the corresponding fluorescent or chemiluminescent secondary antibody. Then, a suitable detection system is used for development.

 

3. Result Analysis 

Ubiquitinated proteins are represented as higher molecular weights than the target protein in Western blot. The level of protein ubiquitination can be estimated by comparing the ubiquitin signal with the target protein signal.

 

Detecting the level of protein ubiquitination requires a deep understanding of sample treatment and the specific protein's ubiquitination status. Additionally, such experiments require high-quality antibodies and optimized experimental conditions to obtain repeatable and accurate results.