P4HB Inhibitor Screening Assay Kit (ab139480)
Key features and details
- Assay type: Enzyme activity
- Detection method: Fluorescent
- Platform: Microplate reader
- Sample type: Inhibitor compounds, Purified protein
- Sensitivity: 37 µM
Overview
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Product name
P4HB Inhibitor Screening Assay Kit
See all P4HB kits -
Detection method
Fluorescent -
Sample type
Purified protein, Inhibitor compounds -
Assay type
Enzyme activity -
Sensitivity
37 µM -
Product overview
Abcam's P4HB (PDIA1) Inhibitor Screening Assay Kit (ab139480) provides a simple, homogenous assay for screening modulators of protein disulfide isomerase (PDIA1) enzymatic activity in a 96-well microplate. The basis for the assay is the P4HB (PDIA1)-catalyzed reduction of insulin in the presence of DTT, resulting in the formation of insulin aggregates which in turnn bind to the red-emitting fluorogenic P4HB (PDIA1) Detection Reagent.
Relative to analogous turbidimetric assays of P4HB (PDIA1) activity, the fluorescence-based assay provides a vastly improved assay signal window, improved lower detection limit and superior Z’-score (>0.8).
The kit includes human recombinant P4HB (PDIA1) enzyme as positive control, as well as the P4HB (PDIA1) inhibitor bacitracin as a negative control.
This kit will provide a quantitative readout of P4HB (PDIA1) enzymatic activity is a robust and HTP fashion and can be applied to identification of P4HB (PDIA1) inhibitors from chemical libraries.
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Platform
Microplate reader
Properties
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Storage instructions
Please refer to protocols. -
Components 2 x 96 tests Deionized Water 1 x 5ml DTT 2 x 1.3ml Inhibitor Control (Bacitracin) 1 vial Insulin (from bovine pancreas) 2 vials PBE Buffer 1 x 25ml P4HB (PDIA1) (Human, Recombinant) 2 x 165µl P4HB (PDIA1) Detection Reagent 1 x 20µl Stop Reagent 1 x 1ml -
Research areas
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Function
This multifunctional protein catalyzes the formation, breakage and rearrangement of disulfide bonds. At the cell surface, seems to act as a reductase that cleaves disulfide bonds of proteins attached to the cell. May therefore cause structural modifications of exofacial proteins. Inside the cell, seems to form/rearrange disulfide bonds of nascent proteins. At high concentrations, functions as a chaperone that inhibits aggregation of misfolded proteins. At low concentrations, facilitates aggregation (anti-chaperone activity). May be involved with other chaperones in the structural modification of the TG precursor in hormone biogenesis. Also acts a structural subunit of various enzymes such as prolyl 4-hydroxylase and microsomal triacylglycerol transfer protein MTTP. -
Sequence similarities
Belongs to the protein disulfide isomerase family.
Contains 2 thioredoxin domains. -
Cellular localization
Endoplasmic reticulum lumen. Melanosome. Cell membrane. Highly abundant. In some cell types, seems to be also secreted or associated with the plasma membrane, where it undergoes constant shedding and replacement from intracellular sources (Probable). Localizes near CD4-enriched regions on lymphoid cell surfaces. Identified by mass spectrometry in melanosome fractions from stage I to stage IV. - Information by UniProt
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Alternative names
- Cellular thyroid hormone binding protein
- Cellular thyroid hormone-binding protein
- Collagen prolyl 4 hydroxylase beta
see all
Images
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Assay validation using Inhibitor Control (bacitracin).
Dose response assay was performed with 0 to 3000 µM bacitracin added 15 min prior to the initiation of enzymatic reaction. Reactions were performed as described in Assay Protocol section. The fluorescence-based assay provides a vastly improved assay signal window and improved lower detection limit, In addition, the Z’-factor score obtained using the assay (0.91 for assay with and without P4HB) demonstrates excellent signal-to-noise and signal-to-background ratio
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Intra-plate and inter-plate reproducibility using Inhibitor Control (bacitracin)Dose response assay was performed with 0 to 3000 µM bacitracin added 15 minutes prior to the initiation of enzymatic reaction. Reactions were performed as described in Methods and Procedures section. Intra-plate and inter-plate CVs using the assay are typically 3-6%.
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Absorption and fluorescence emission spectra for P4HB (PDIA1) Detection Reagent.All spectra were determined in PBE Buffer. Example data only.