Ubiquitination is a process in which Ubiquitin molecules specifically bind to, and mark, proteins for degradation. There are traditionally three mechanisms through which ubiquitin can be quantified; in which the ubiquitin is cleaved off of the protein marked for degradation and then measured. In which ubiquitin is immunoprecipitated from a cell lysate. In which reactions catalyzing ubiquination are measured using biotinylated-protein substrate
Ubiquitin is a small protein that is attached to proteins to mark them for degradation. Ubiquitination is a highly regulated process in which Ubiquitin molecules specifically bind to, and mark, proteins for degradation.
The traditional methods of measuring ubiquitination are:
This method is done by sandwiching a polypeptide onto the surface of nitrocellulose or PVDF membranes, which are then incubated with different antibodies that target specific sites on the protein. After a wash to remove non-specific interactions, another antibody against an epitope on the first antibody is added and incubated again. The resulting product can be detected using chemiluminescence, immuno-gold staining, colorimetric reaction or autoradiography.
The ubiquitin is cleaved off of the protein marked for degradation and then measured.
This can be done by enzymatic methods that cleave ubiquitin from its target protein, or by any other method that allows quantification of the amount of ubiquitin present.
To perform this experiment, cells are treated with an agent that makes ubiquitin proteins accessible to the immune system. The immune system then binds to these proteins with antibodies, which are then pulled out of the cell lysate (the solution containing all of the cell's contents).
Here's a quick look at how the ubiquitination reaction is measured in the lab:
A fourth mechanism using Surface Plasmon Resonance spectroscopy has also been developed to directly quantify the affinity of ubiquitin and its targets. The technique involves coupling a protein of interest with a gold surface, which acts as a mirror that reflects light back at an angle depending on how strongly the two interact.
Ubiquitination is a process in which Ubiquitin molecules specifically bind to, and mark, proteins for degradation. There are traditionally three mechanisms through which ubiquitin can be quantified;
Determining the level of ubiquitinated protein in a cell or tissue sample is typically done by immunoblotting, using a ubiquitin-specific antibody. The antibody binds to its target and then a secondary antibody conjugated to a reporter molecule can be used to detect it. This can be detected quantitatively using an enzyme-linked immunosorbent assay (ELISA).
Immunofluorescence and immunohistochemistry can be performed on tissues directly without the need for blotting. Immunoblotting requires a gel to separate the proteins, which often results in multiple bands for each protein. The ubiquitin-specific antibodies will not bind to other proteins so it is easier to see where your sample shows up on the immunoblotting gel.
An alternative, but less sensitive, method is based on the use of ubiquitin-proteasome inhibitors such as MG132 or epoxomicin to block proteosomal degradation and accumulate polyubiquitinylated proteins in cells. This method can be used to study the role of ubiquitination in a variety of processes including apoptosis and cell division.
Proteasome inhibitors like MG132 and lactacystin are frequently used to enrich for polyubiquitinylated proteins from cell lysates, since the proteasome inhibitors may also stabilize other forms of protein conjugates such as multi-protein complexes and phosphorylated proteins.
In addition to immunoblotting and immunofluorescence, there are a variety of methods that can be used to quantify ubiquitination. For example, you can quantify ubiquitination levels in cells by using immunohistochemistry. In this method, antibodies that recognize specific epitopes on proteins tagged with ubiquitin will bind to their targets and indicate the presence of these tagged proteins in your sample. There are also several different types of proteasome inhibitors available that can be used to assess whether or not an enzyme involved in protein degradation is upregulated due to high levels of ubiquitination.
To summarize, in order to accurately measure ubiquitin levels in your sample, you need to determine the specific method that works best for your assay. In general, the most common way is by using Western blotting or mass spectrometry. These methods are widely used because they have been proven reliable over time and because they can be performed quickly (which is important when working with live cells).