Peptide Exchange Assay for HLA-E

HLA-E-β2m complexes previously refolded with the UV-sensitive VL9 peptide (VMAPJTVLL) were incubated in the presence of molar excess test peptide and evaluated via the Blue Native-PAGE Novex Bis–Tris gel system (life technologies) (Walters et al., 2018 (link)). In brief, pre-refolded and purified HLA-E in complex with the UV-sensitive peptide was incubated for 3 h on ice in the presence of 12 M excess test peptide prior to the addition of 3 μL 4× Native-PAGE Sample Buffer per 10 μg (10 μL) of sample. Samples were loaded onto 4–16% Native-PAGE Novex Bis–Tris gels with NativeMark Unstained Protein Standard used as the ladder control. Gel electrophoresis was carried out at 150 Volts for 2 h at RT with a current gradient ranging from 15 to 16 to 2–4 mAmps. Gels were subsequently rinsed three times in MilliQ water and stained for 2–3 h in SimplyBlue SafeStain at RT. The MiliQ water was changed a number of times over a 24–48 h period to enable gel de-staining.

Free full text: Click here

Walters L.C., Rozbesky D., Harlos K., Quastel M., Sun H., Springer S., Rambo R.P., Mohammed F., Jones E.Y., McMichael A.J, & Gillespie G.M. (2022). Primary and secondary functions of HLA-E are determined by stability and conformation of the peptide-bound complexes. Cell Reports, 39(11), 110959.

Publication 2022

Blue Native-PAGE Novex Bis–Tris gel system

Bis tris Buffer Electrophoresis Hla e Molar Native page Peptide Peptide m Protein

Corresponding Organization : University of Oxford

Other organizations : Charles University, Wellcome Centre for Human Genetics, First Hospital of China Medical University, China Medical University, Constructor University, Diamond Light Source, University of Birmingham

Top 5 similar protocols

Variable analysis

independent variables

Presence of test peptide (molar excess)

dependent variables

Dissociation/exchange of UV-sensitive VL9 peptide from pre-refolded and purified HLA-E complexes, as observed via Blue Native-PAGE

control variables

Pre-refolded and purified HLA-E in complex with the UV-sensitive VL9 peptide
Incubation time (3 hours)
Incubation temperature (on ice)
Volume of 4× Native-PAGE Sample Buffer added per 10 μg (10 μL) of sample
Gel system (4–16% Native-PAGE Novex Bis–Tris gels)
Electrophoresis conditions (150 Volts for 2 h at RT with a current gradient ranging from 15 to 16 to 2–4 mAmps)
Staining and de-staining procedures (SimplyBlue SafeStain, 2–3 h at RT, multiple rinses in MilliQ water over 24–48 h period)

controls

Positive control: None mentioned
Negative control: None mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!