Cullin4a/Rbx1/DDB1/CRBN E3 Ligase Complex TR-FRET Kit

32160000

Aliquot to avoid freeze/thaw cycles.Avoid freeze/thaw cycles.

100x UBA1 (150µl) | 100x UBE2D1 (150µl) | 100x Cul4a/Rbx1 Complex (neddyl.) (150µl) | 100x DDB1/CRBN (150µl) | 10x Reaction Buffer (1ml) | 10x Mg-ATP (1ml) 1 x 384-well white low-volume microplate

South Bay Bio?s Cullin4a/Rbx1/DDB1/CRBN Neddylated E3 Ligase TR-FRET Kit provides a fast and sensitive method monitoring ubiquitin conjugation onto the E3 complex in solution, resulting from an enzymatic ubiquitin cascade without the need of running and staining an SDS gel. The kit enables continuous TR-FRET detection of ubiquitin chain formation onto CRBN (or substrates) in a real-time detection setup, or in an end-point configuration if desired. TR-FRET uses the extended fluorescence emission decay lifetimes typical of rare-earth lanthanides to impart a short time-delay between FRET donor excitation and emission. This delay provides a means to separate ?true? signal from short-lived background fluorescence, and reduce interference from compound fluorescence and other assay artifacts.

The kit uses ubiquitin labeled with either Europium-Cryptate or Cy5 as FRET pair donor and acceptor fluorophores respectively, completely eliminating the need for antibody based detection setups. Enzymatic incorporation of the labeled ubiquitins into chains conjugated onto CRBN (or substrates) leads to an increase in fluorescence emission at 665nm (Em-Acceptor) and decrease at emission wavelength 620nm (Em-Donor).

Vial

South Bay Bio?s Cullin4a/Rbx1/DDB1/CRBN Neddylated E3 Ligase TR-FRET Kit provides a fast and sensitive method monitoring ubiquitin conjugation onto the E3 complex in solution, resulting from an enzymatic ubiquitin cascade without the need of running and staining an SDS gel. The kit enables continuous TR-FRET detection of ubiquitin chain formation onto CRBN (or substrates) in a real-time detection setup, or in an end-point configuration if desired. TR-FRET uses the extended fluorescence emission decay lifetimes typical of rare-earth lanthanides to impart a short time-delay between FRET donor excitation and emission. This delay provides a means to separate ?true? signal from short-lived background fluorescence, and reduce interference from compound fluorescence and other assay artifacts.

Readout: Endpoint/Kinetic Label or Dye: Europium-Cryptate & Cy5 Detection Method: TR-FRET Substrate Properties: Protein-based Substrate. Typical experimental concentration is 1x. Excitation/Emission (nm): 304/620 & 304/665

Non-hazardous

Chemistry test kits or supplies

41116104