Dcaf11-Driven p21 Ubiquitination Assay Kit

Catalog #

78892

$1,450 *

Size: 384 reactions

Qty

Bulk/Custom

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Description

The Dcaf11-driven p21 ubiquitination assay kit is a sensitive AlphaLisa® high-throughput screening (HTS) assay kit, designed to measure Dcaf11 (DDB1- and Cul4-associated factor 11) E3 ligase activity in a homogeneous 384 reaction format. The assay kit comes with enough biotinylated ubiquitin, ATP, GST-tagged p21, assay buffer, detection buffer, purified UBE1 (E1), UbcH5b (E2), and Rbx1/Cul4B/Dcaf11/Ddb1 complex (E3) for 384 reactions. The assay detects mono-ubiquitination and poly-ubiquitination of p21.

Figure 1. Dcaf11-driven p21 ubiquitination assay kit schematic.
First, E1 and E2 enzymes are incubated with the E3 complex and with the target protein p21 (GST-tag) in the presence of biotin-conjugated ubiquitin and ATP. Ubiquitination of p21 occurs in a multistep ubiquitin transfer from E1 to E2 to E3, and E3-mediated conjugation of ubiquitin to p21. Next, glutathione-acceptor beads are added, followed by streptavidin-conjugated donor beads, and Alpha-counts are measured. The increase in Alpha-counts is proportional to the mono- or poly-ubiquitination of the GST-tagged p21.

Need us to run inhibitor screens or profile your compounds against Dcaf11-Driven p21? Check out our Ubiquitination Screening Services.

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Synonyms

ubcH5b, UBE2D2, UBE1, UBA1, DNA damage-binding protein 1, XAP1, DDB1- and CUL4-associated factor 11, WD repeat-containing protein 23, WDR23, GL014, PRO2389, KIAA0695, Cullin-4B, RNF75, ROC1, A1S9T, UBE1, PUBC1, UBC4, UBC5B, UBCH4, UBCH5B, DCAF-11

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Product Data Gallery

Dcaf11-driven p21 ubiquitination

Product Info

Storage and Usage

Citations

Assay Kit Format

AlphaLISA®

Materials Required But Not Supplied

AlphaLISA® glutathione acceptor beads (Perkin Elmer #AL109C)
AlphaScreen® Streptavidin-conjugated donor beads (Perkin Elmer #6760002S)
Optiplate-384 (Perkin Elmer #6007290)
AlphaScreen® microplate reader
DNAse free water

Format

Catalog #	Name	Amount	Storage
80301	UBE1 (UBA1), Flag-Tag Recombinant*	25 µg	-80°C
80314	UbcH5b, His-Tag (Human) Recombinant*	50 µg	-80°C
101495	Rbx/Cul4B/Dcaf11/Ddb1 Complex*	5 µg	-80°C
101584	p21, GST-Tag*	5 µg	-80°C
	Biotin-Ubiquitin	400 µl	-80°C
	ATP (10 mM)	400 µl	-80°C
	U2 Assay Buffer	2 x 10 ml	-80°C
	4x U2 Detection Buffer	2 x 2 ml	-20°C

*The initial concentration of enzyme is lot-specific and will be indicated on the tube containing the protein.

UniProt #

UBE1: P22314; UbcH5b: P62837; Rbx1: P62877; Cul4B: Q13620; Dcaf11: Q8TEB1; Ddb1: Q16531

Background

Covalent conjugation to ubiquitin (Ub) is one of the major post-translational modifications regulating protein stability, function, and localization. Ubiquitination is the concerted action of three enzymes: a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2), and a Ub ligase (E3). The specificity and efficiency of ubiquitination are largely determined by the E3 enzyme, which directs the last step of the Ub-conjugating cascade by binding to both an E2∼Ub conjugate and a substrate protein. This step ensures the transfer of Ub from E2∼Ub to the substrate, leading to its mono- or poly-ubiquitination.

DDB1 and CUL4-associated Factor 11 (Dcaf11, also known as WD Repeat Domain 23, WDR23) is a protein that associates with Cul4A or Cul4B to form a complex with E3 ligase activity. Dcaf11 E3 ligase can ubiquitinate a variety of substrates, including p21 (cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1). P21 is involved in cell cycle regulation by inhibiting CDK (cyclin-dependent kinase) and PCNA (proliferating cell nuclear antigen). It also mediates p53 response to DNA damage, and its levels have been linked to lymph node metastasis and overall survival rate to cancer. Knocking down any component of the Dcaf11 complex attenuates the ubiquitination level of p21, inhibiting osteosarcoma cell proliferation. The use of drugs targeting the Dcaf complex may be a viable cancer therapy approach.

Storage/Stability

This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.

Applications

Screen molecules that inhibit Dcaf11 Ub ligase activity in drug discovery high throughput screening (HTS) applications.
Determine compound IC₅₀
Analyze Dcaf11-driven ubiquitination.

Shipping Temperature

-80°C

Notes

Troubleshooting Guide
Visit bpsbioscience.com/assay-kits-faq for detailed troubleshooting instructions. For all further questions, please email [email protected]

Contraindications

The Dcaf11-driven p21 Ubiquitination Assay Kit is compatible with up to 1% final DMSO concentration. We recommend preparing the inhibitor in no higher than 4% DMSO solution in buffer and using 2.5 µl per well.
Green and blue dyes that absorb light in the AlphaScreen® signal emission range (λ=520-620 nm), such as Trypan Blue, interfere with the assay.
Avoid the presence of potent singlet oxygen quenchers such as sodium azide (NaN₃) or metal ions (Fe²⁺, Fe³⁺, Cu²⁺, Zn²⁺ and Ni²⁺).
The presence of the culture medium RPMI 1640 at >1% leads to signal reduction due to the presence of excess biotin and iron in this medium. MEM, which lacks these components, does not affect AlphaScreen® assays.

Warnings

Avoid freeze/thaw cycles

Disclaimers and Limitations

BPS Bioscience assay kits are validated using the listed components according to our specific protocol. Any deviations to kit components or protocols, such as using different proteins, cell/tissue lysates, or buffers (including using the same component from other commercial sources) will void any warranties on the performance of the assay kit and is not recommended.

End-users should immediately report any issues to [email protected] upon receipt of the kit, such as missing components, damaged vials, no dry ice, etc.

Scientific Category

Ubiquitination

Regulatory Status

Research Use Only