Ubiquitinylation kit

Short Name :

Ubiquitinylation kit

Cat.No. :

KITZ-010

Size :

50 x 50 µl reactions

Product Overview :

Ubiquitinylation kit provides the means of generating a range of thioester-linked ubiquitin conjugation enzymes (E2s), utilizing the first two steps in the ubiquitin cascade; for use in the transfer of ubiquitin to E3 ligases; and the subsequent ubiquitinylation of target proteins. Biotinylated ubiquitin provided supports thioester formation and high sensitivity detection of ubiquitin conjugates. Suggested uses: Generation of ubiquitin-E2 thioesters for use in ubiquitinylation experiments, Ubiquitinylation of target proteins in the presence of a dedicated E3 ligase, Activation of ubiquitin for thioester conjugation to novel E2 enzymes, Use of cell lysate or crude fractions/preparations as source of E3 ligases to facilitate ubiquitinylation, Substrate (target) independent in vitro ubiquitinylation reactions.

Applications :

For generation of ubiquitin-E2 thioesters for use in ubiquitinylation experiments.

Storage :

All kit components should be stored at -80°C to ensure stability and activity.

Shipping :

Dry ice

Kit Components :

1. 20x Ubiquitin Activating Enzyme Solution (E1): 1 vial
E1 (human) (recombinant) (His-tag), Use 2.5 µL per 50 µL reaction. 125 µL provided, sufficient for 50 x 50 µL reactions.
2. 10x Ubiquitin Conjugating Enzyme Solutions (E2): 11 vials:
UbcH1 (human), (recombinant) (His-tag)
UbcH2 (human), (recombinant) (His-tag)
UbcH3 (human), (recombinant) (His-tag)
UbcH5a (human), (recombinant) (His-tag)
UbcH5b (human), (recombinant) (His-tag)
UbcH5c (human), (recombinant) (His-tag)
UbcH6 (human), (recombinant) (His-tag)
UbcH7 (human), (recombinant) (His-tag)
UbcH8 (human), (recombinant) (His-tag)
UbcH10 (human), (recombinant) (untagged)
UbcH13/Mms2 (human), (recombinant) (His-tag)
Use 5 µL per 50 µL reaction. 20 µL of each E2 provided, sufficient for 4 x 50 µL reactions.
Note: UbcH2 is sensitive to reducing agents such as DTT and BME. Do not use reducing agents with this enzyme. Assays performed with this enzyme must use only nonreducing buffers.
3. 20x Biotinylated Ubiquitin Solution (Bt-Ub): 1 vial:
Biotinylated-ubiquitin. Use 2.5 µL per 50 µL reaction, 125 µL provided, sufficient for 50 x 50 µL reactions.
4. 20x Mg-ATP Solution: 1 vial:
Mg-ATP. Use 2.5 µL per 50 µL reaction, 125 µL provided, sufficient for 50 x 50 µL reactions.
5. 2x Non-reducing Gel Loading Buffer:
2 vials of 1.25 mL, Use 50 µL per 50 µL reaction. 2.5 mL provided, sufficient for 50 x 50µL reactions
6. 10x Ubiquitinylation Buffer: 1 vial:
Use 5 µL per 50 µL reaction, 250 µL provided, sufficient for 50 x 50 µL reactions.