Transcreener ADP² Assay directly measures the ADP produced by VPS4A (Vacuolar protein sorting-associated protein 4A). The ADP measurements allow researchers to effectively determine the activity of the VPS4A enzyme. The assay provides a powerful tool for screening compound libraries for VPS4A modulators to help find new therapies for disease.
The kit includes the detection reagents required to measure activity. VPS4A enzyme is not included in the inhibitor screening assay kit but can be purchased through BellBrook Labs.
Looking for a detailed protocol for performing the assay in your lab? Download the application note to get the assay up and running in your lab within hours.
Run your enzyme reaction, add Transcreener reagents, and read your plates. The VPS4A assay is compatible with 96, 384, and 1536-well formats.
Z’ measurements using optimized VPS4A reaction conditions indicate a robust assay. Robust data like this is vital for sizeable, high-throughput screens that are difficult to complete due to the massive sample quantity. Z’ shown here = 0.91.
The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here, we use 2 µM ATP (sub-Km concentration) in a reaction buffer containing 50 mM HEPES (pH 7.5), 10 mM MgCl2, 50 mM KCl, 5 mM DTT, and 0.01% Triton at 37°C for two hours. Linearity is shown here under initial velocity conditions when raw data is converted to ADP formed.
Dose-response testing of selected hits from the pilot screen (BVT948 and Polygodial) and the control inhibitor (DBeQ) against VPS4A (175nM).
Interested in moving your program forward but don’t want to bring an assay in-house? Our scientists can help! BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your AAA ATPase program.
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| Component | Notes |
|---|---|
| ADP² Antibody | Antibody specific to the nucleotide ADP. |
| ADP Tracer | When displaced, changes in fluorescence can be read in an FP, FI, or TR-FRET depending on the assay format. |
| Stop & Detect Buffer B, 10X for FP & FI, Stop & Detect Buffer C, 10X for TR-FRET | 200 mM HEPES (pH 7.5), 400 mM EDTA, and 0.2% Brij-35. Stops enzyme reactions requiring Mg²+. |
| ATP and ADP | ATP and ADP are used to create the ATP/ADP standard curve. ATP is used as a phosphate donor to start the reaction. |
VPS4A (Vacuolar Protein Sorting 4A) is a crucial protein that plays an essential role in the endosomal sorting complexes required for the transport (ESCRT) pathway. This pathway is vital for various cellular processes, including the sorting of membrane proteins, vesicle trafficking, and the formation of multivesicular bodies (MVBs). VPS4A belongs to the AAA ATPase family and is key to disassembling the ESCRT-III complex, which is critical for the final stages of endosomal sorting, as well as for processes such as cytokinesis, viral budding, and autophagy.
Given VPS4A’s involvement in these biological functions, it presents a promising therapeutic target for a range of diseases, including cancer, viral infections, and neurodegenerative disorders. Ongoing research into the inhibition or modulation of VPS4A may lead to novel therapeutic approaches that could improve existing treatments or create new methods to tackle difficult diseases.
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