Figure 1. Poly-ubiquitination vs Multi-mono-ubiquitination |
Ubiquitin can be attached to a protein substrate via two distinct mechanisms (Figure 1). Poly-ubiquitination occurs when Ubiquitin molecules are attached end-to-end to a single lysine residue on a substrate protein to form a poly-ubiquitin chain (Figure 1A). Alternatively, multi-mono-ubiquitination is the attachment of a single Ubiquitin molecule to multiple lysine residues on a substrate protein (Figure 1B). It is important to distinguish between poly-ubiquitination and multi-mono-ubiquitination because these different types of ubiquitination lead to different functions of the substrate protein. To complicate matters, a poly-ubiquitinated protein and a multi-mono-ubiquitinated protein look very similar by SDS-PAGE and Western blot. Fortunately, it is relatively simple to differentiate between poly-ubiquitination and multi-mono-ubiquitination by performing in vitro ubiquitin conjugation reactions. The protocol below describes in detail how to determine if your protein of interest is poly-ubiquitinated or multi-mono-ubiquitinated. Click on the links below to view listings of Boston Biochem® proteins, enzymes, and buffers.
Material or Reagent | Stock Concentration |
E1 Enzyme | 5 µM |
E2 Enzymei | 25 µM |
E3 Ligaseii | 10 µM |
10X E3 Ligase Reaction Buffer | 10X - (500 mM HEPES, pH 8.0, 500 mM NaCl, 10 mM TCEP) |
Ubiquitin | 1.17 mM (10 mg/mL) |
Ubiquitin No K | 1.17 mM (10 mg/mL) |
MgATP Solution | 100 mM |
SDS-PAGE sample buffer – if not using reaction products for downstream applications | 2X |
EDTA or DTT – if using reaction products for downstream applications | 500 mM (EDTA);1 M (DTT) |
Microcentrifuge tubes | |
Water Bath (37 °C) | |
Western Blot Equipment |
iEach E2 enzyme functions with only a subset of E3 ligases and some E3’s are more promiscuous than others.
iiThe E3 ligase will likely need to be supplied by the user, but we do offer a small selection.
Figure 2. Wild Type Ubiquitin vs Ubiquitin No K |
Two in vitro Ubiquitin conjugation reactions will need to be performed: 1) one that contains wild type Ubiquitin and 2) one that contains Ubiquitin No K, a mutant in which all 7 lysines have been mutated to arginines. Wild type Ubiquitin can be conjugated to substrate proteins and is capable of forming chains (Figure 2; left). Ubiquitin No K can also be conjugated to substrate proteins, but it is unable to form chains due to its lack of lysine residues (Figure 2; right). Therefore, if your substrate is poly-ubiquitinated, high molecular weight bands will be generated in reaction 1, but not reaction 2 (Figure 3A). Conversely, if your substrate is multi-mono-ubiquitinated, high molecular weight bands will be generated in both reaction 1 and reaction 2 (Figure 3B). Note that reaction 1 will look the same for both poly-ubiquitinated and multi-mono-ubiquitinated substrates.
Figure 3. Differentiation Between Poly-Ubiquitination and Multi-Mono-Ubiquitination Using Ubiquitin No K |
Reagent | Volume | Working Concentration |
dH2O | X µL (to 25 µL; dependent on volume of substrate and E3 ligase) | N/A |
10X E3 Ligase Reaction Buffer | 2.5 µL | 1X - (50 mM HEPES, pH 8.0, 50 mM NaCl, 1 mM TCEP) |
Ubiquitin | 1 µL | Approximately 100 µM |
MgATP Solution | 2.5 µL | 10 mM |
Substrate | X µLiii | 5-10 µM |
E1 Enzyme | 0.5 µL | 100 nM |
E2 Enzyme | 1 µL | 1 µM |
E3 Ligase | X µLiv | 1 µM |
iiiThe volume needed will depend on the stock concentration of your substrate.
ivThe volume needed will depend on the stock concentration of your E3 ligase.
Reagent | Volume | Working Concentration |
dH2O | X µL (to 25 µL; dependent on volume of substrate and E3 ligase) | N/A |
10X E3 Ligase Reaction Buffer | 2.5 µL | 1X - (50 mM HEPES, pH 8.0, 50 mM NaCl, 1 mM TCEP) |
Ubiquitin No K | 1 µL | Approximately 100 µM |
MgATP Solution | 2.5 µL | 10 mM |
Substrate | X µLiii | 5-10 µM |
E1 Enzyme | 0.5 µL | 100 nM |
E2 Enzyme | 1 µL | 1 µM |
E3 Ligase | X µLiv | 1 µM |
iiiThe volume needed will depend on the stock concentration of your substrate.
ivThe volume needed will depend on the stock concentration of your E3 ligase.
Are you using reaction products for downstream enzymatic applications? | Termination method | Volume (final concentration) |
No | SDS-PAGE sample buffer | 25 µL (1X) |
Yes | EDTA or DTTv | 0.5 µL EDTA (20 mM) or 1 µL DTT (100 mM) |
vEDTA and DTT are equally effective at terminating the reaction; determining which to use will depend on the intended downstream enzymatic application of the reaction products.
*It is possible for a substrate to be both poly-ubiquitinated and multi-mono-ubiquitinated. If this is the case the Ubiquitin No K will still prevent poly-ubiquitination and the highest molecular weight protein species should disappear from reaction 2. Contact us for technical assistance!