Ubiquitin Ligases
This class of ubiquitin ligases, which was discovered in 1999, has a signature domain that was first identified within a "really interesting new gene" called RING1.
What are RING E3s?
To induce the expression of a recombinant protein in E. coli, we add this chemical to the culture, which binds to the lac repressor to trigger expression of the target protein.
What is IPTG?
This lysine residue of ubiquitin is the only essential lysine in budding yeast, consistent with the role of chains linked through this site in degradation by the proteasome.
What is K48?
This general protein stain binds primarily to basic residues and should be handled carefully (Anthony) to avoid unsightly stains in the lab.
What is Coomassie stain?
A role for this protein in proteolysis was first described in 1980, when Hershko and Ciechanover discovered a heat-stable protein in reticulocytes called APF-1 that regulates ATP-dependent degradation.
What is ubiquitin?
In humans, there are predicted to be approximately this many HECT ubiquitin ligases, although not all of them have been demonstrated yet to have ubiquitin ligase activity.
What is 28-30?
The 10x reaction buffer that we use for ubiquitination assays contains this critical component, without which ubiquitin would not be able to be activated by E1.
What is ATP?
Ubiquitin chains linked through this lysine residue are involved in proteasome-independent processes and have an "open" or extended conformation in solution.
What are K63-linked chains?
Sterile tips and tubes should be incubated in this heated chamber immediately after autoclaving to remove condensation and to prevent warping of plastic.
What is the drying oven?
A chain containing a minimum of this number of ubiquitin molecules linked through K48 was first described as the canonical signal for protein degradation by the proteasome in 1989.
What is four?
The founding member of this class of ubiquitin ligases is frequently mutated in Parkinson's disease and works through a RING-HECT hybrid catalytic mechanism.
What are RBR ubiquitin ligases?
To purify recombinant proteins, we generally use this type of chromatography, which is based on the ability of a protein tag to bind to an immobilized ligand.
What is affinity chromatography?
These types of ubiquitin chains contain at least one ubiquitin molecule that is simultaneously modified on multiple acceptor sites and have the ability to act as powerful degradation signals.
What are branched ubiquitin chains?
In line with American flag t-shirts, this Dr. C. and possibly QAnon inspired door code is used for access to the research lab from the teaching lab and vice versa.
What is 1776?
Avram Hershko, Aaron Ciechanover, and Irwin Rose won the Nobel Prize in 2004 in this field for their discovery of ubiquitin-mediated proteolysis.
What is chemistry?
This gene is among the most frequently mutated in breast cancer and its protein product forms a ubiquitin ligase complex with BARD1 to modify substrates.
What is BRCA1?
In SDS-PAGE, this critical reagent is added to the sample buffer to ensure that all protein cysteine residues are reduced during electrophoresis.
What is mercaptoethanol (BME)?
Ubiquitin chains linked through this lysine residue have been implicated in the DNA damage response and mitophagy, and they have a closed conformation similar to that of K48-linked chains.
What are K6-linked chains?
This metal and wood animal-catching device once had its home in the fume hood but has since been replaced by an orbital shaker and a sonicator.
What is a rat trap?
This K48-linked chain forming HECT E3 was the first ubiquitin ligase to be discovered in 1995 and binds to the E6 protein in HPV infected cells to trigger p53 degradation.
What is E6AP?
LUBAC is a multi-protein complex and currently the only ubiquitin ligase known to form chains through this acceptor site of ubiquitin.
What is M1 (N-terminal methionine)?
In western blotting, the secondary antibodies that we use are conjugated to this enzyme, which forms a product that give off light to enable the detection of protein bands.
What is horseradish peroxidase?
Ubiquitin chains linked through this lysine have an important role in the cell cycle and are synthesized by the anaphase promoting complex in conjunction with the E2 enzyme, UBE2S.
What are K11-linked chains?
This Cass door code, which is required to access the ice machine, is also the year that K48-linked ubiquitin chains was first described as the canonical signal for proteasomal degradation.
What is 1989?
This DNA binding protein was the first protein reported to be ubiquitinated in 1977 and remains one of the best examples of a protein that is regulated by monoubiquitination.
What is histone H2A?