Chromogenic Polymer Hydrogel-Based Enzyme Screening Assay: A High Throughput Method to Screen the Carbohydrate-Active Enzymes Using Synthetic Substrates
Published: April 30, 2023
Abstract
Source: Schückel, J., et al. High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits. J. Vis. Exp. (2016).
In this video, we demonstrate a high throughput assay for carbohydrate-active enzyme screening using insoluble chromogenic polymer hydrogel substrates. The enzyme degrades these substrates into a soluble colored product that can be quantified by measuring the color intensity of the product.
Protocol
1. Activation of the assay kit plate Activate the 96-well filter (containing blue CPH-xylan) assay kit plate (List of Materials) by adding 200 µl activation solution (obtained with the kit) into each well, followed by 10 min incubation at room temperature without agitation. Apply vacuum using a vacuum manifold (with the spacer block inside and any standard, transparent 96-well plate as a collection plate) to remove the extant activation solution. It is also possible to use a ce…
Divulgaciones
The authors have nothing to disclose.
Materials
| assay kit plates | Glycospot | customized assay kit plates | |
| activation solution | Glycospot | for activating CPH substrates | |
| 350 ml receiver plate spacer block for vacuum manifold | Pall Corporation | 5015 | spacer block |
| 96-well MultiScreen HV filter plate, 0.45 µm, clear, non-sterile | Millipore | MSHVN4510 | assay plate |
| 96-Well Microplates, Polypropylene | Greiner Bio-One | 651201 | collection plate after washing the substrates |
| Nunc MicroWell 96-Well Microplates | Thermo Scientific | 269620 | product plate |
| Diaphragm pump MZ 2 NT | Vacuubrand | 732000 | vacuum pump used with the vacuum manifold |
| Infors HT Ecotron | Infors HT | 4950132 (Buch & Holm) | horizontal shaker |
| SpectraMax M5 | Molecular Devices | 10067-750 (VWR) | 96-well plate absorbance reader |
| Vacuum manifold | Pall Corporation | 5017 | vacuum manifold |
| endo-cellulase (EGII) (Trichoderma longibrachiatum) | Megazyme | E-CELTR | cellulase [cel] |
| α-amylase (Bacillus licheniformis) | Megazyme | E-BLAAM | amylase [amy] |
Tags
Citar este artículo
Chromogenic Polymer Hydrogel-Based Enzyme Screening Assay: A High Throughput Method to Screen the Carbohydrate-Active Enzymes Using Synthetic Substrates. J. Vis. Exp. (Pending Publication), e21176, doi: (2023).