Skip to main content
Menu
US

HTRF Human and Mouse Total Huntingtin (HTT) Detection Kit, 500 Assay Points

The HTRF Total Huntingtin kit is designed for the simple and rapid quantification of soluble total HTT (WT & mutant forms) in cell/tissue lysates.

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption & disposal requirements under European REACH regulations (EC 1907/2006).

Product Variants
Part number: 64HTTTPEG
Unit Size: 500 Assay Points
List price: USD 1,604.00
Your price:
USD 1,604.00
USD 1,604.00 /each
Part number: 64HTTTPEH
Unit Size: 10,000 Assay Points
List price: USD 19,730.00
Your price:
USD 0.00
USD 19,730.00 /each

Overview

Huntingtin (HTT) is a cytoplasmic protein which is highly expressed in the brain, and whose anti-apoptotic role is critical for neuronal survival.

The wild-type (WT) protein has a functional structure, with a "normal" polyglutamine (polyQ) domain containing less than 36Q. The mutant HTT protein harbors an abnormally long polyQ tract (> 36Q), which causes the aggregation of the no longer functional protein. HTT aggregation leads to the selective neuronal cell death responsible for Huntington's Disease.

Specifications

Application
Protein Detection
Assay Points
500
Assay Target Type
Kit
Assay Technology
HTRF
Automation Compatible
Yes
Brand
HTRF
Detection Method
HTRF
Experimental Type
In vitro
Therapeutic Area
Neuroscience
Unit Size
500 Assay Points

Video gallery

How it works

Total HTT assay principle

The Total HTT assay is based on a TR-FRET sandwich immunoassay involving two specific antibodies, one labelled with Tb3+ cryptate (donor) and the other with d2 (acceptor). Both antibodies bind to soluble Total HTT (WT & mutant forms), and the donor-acceptor proximity enables a fluorescent TR-FRET signal. The intensity of the signal is directly proportional to the concentration of soluble Total HTT present in the sample (cell lysate or tissue lysate).

human-mouse-total-huntingtin-detection-kit

 

Total HTT assay protocol

The Total HTT assay can be run in a 96- or 384-well low volume white detection plate (20 µL final). As described here, samples (cell/tissue lysates) or standards are dispensed directly into the assay plate for the detection of Total HTT by HTRF® reagents. The antibodies labelled with HTRF fluorophores may be pre-mixed and added in a single dispensing step. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally.

 

human-mouse-total-huntingtin-detection-kit

 

Assay details

Technical specifications of Total HTT kit
Sample size 10 µL
Final assay volume 20 µL
Time to results Overnight at RT
Kit components
Frozen control lysate, detection antibodies, buffers & protocol
LOD & LOQ (in Lysis Buffer #2) 1.6 ng/mL & 6.3 ng/mL
Assay range 6.3 - 750 ng/mL
Species cross-reactivity
Human, mouse, & rat
Assay specificity Soluble wild-type & mutant HTT forms

 

Assay validation

Analysis of brain tissue samples collected from WT and mutant HTT mouse models

The HTRF Total HTT assay was validated on brain tissues collected from premanifest zQ175 mice and wild-type (WT) littermates, the most extensively studied preclinical knock-in mouse model used for Huntington’s disease (Landles C. et al, Brain Commun. 2021; 3(1):fcaa231). The cortex and cerebellum tissues of three WT mice (#1, #2, & #3) and three mutant zQ175 mice (#4, #5, & #6) were lysed following the procedure given in the kit’s package insert. Briefly, a 5% (w/v) tissue homogenate was prepared using ice-cold 1X lysis buffer #2 supplemented with protease inhibitors, and the insoluble fraction of the lysate containing putative mutant HTT aggregates was removed by centrifugation. The supernatants containing soluble HTT proteins were analysed using the HTRF Total HTT assay and the HTRF Mutant HTT assay (Cat# 64HTTMPEG/H). To check that the protein extraction was similar for each sample, the level of the housekeeping protein GAPDH was also measured using the HTRF GAPDH assay (Cat# 64GAPDHPEG/H). To ensure that the detected analyte was assessed at a concentration compatible with the assay’s linear range, the lysates were pre-diluted in 1X lysis buffer #2 supplemented with protease inhibitors just before detection (1/4 for Total HTT assay, 1/8 for Mutant HTT assay, and 1/100 for GAPDH assay).

The levels of GAPDH were similar for all samples (green bars), demonstrating that the tissue lysates contained comparable total protein contents. No signal was obtained with the Mutant HTT assay on samples collected from WT mice, whereas the soluble mutant protein was clearly detected in cortex and cerebellum tissues collected from premanifest zQ175 mice (blue bars). As expected, the soluble total HTT protein (WT and mutant forms) was measured in all lysates (red bars), and its levels correlate well with the small variations observed for the Mutant HTT protein levels.

human-mouse-total-huntingtin-detection-kit

 

human-mouse-total-huntingtin-detection-kit

 

 

Side-by-side comparison of HTRF and Western Blot for the analysis of Mutant and Total HTT in brain tissues

A side-by-side comparison of HTRF Mutant & Total HTT assays and the Western Blot technique was performed on several brain tissues (cortex, hippocampus, and cerebellum) collected from one WT mouse and one premanifest zQ175 mouse. Tissue lysates were prepared as described in the previous section and the supernatants containing soluble HTT proteins were analysed, either by HTRF using the HTRF Mutant & Total HTT assays, or by Western Blot. To ensure that the detected analyte was assessed at a concentration compatible with the assay’s linear range for each detection method, the lysates were pre-diluted in 1X lysis buffer #2 supplemented with protease inhibitors just before detection (for HTRF: 1/8 for Mutant HTT assay and 1/4 for Total HTT assay; for Western Blot: 1/20 for both assays).

The results obtained using HTRF Mutant & Total HTT assays are comparable with those obtained by Western Blot. With both methods, no soluble mutant HTT was detected in brain tissues collected from WT mice, whereas the protein was properly measured in all samples collected from premanifest zQ175 mice. As expected, the soluble total HTT protein (WT and mutant forms) was measured in all lysates, and its levels correlate well with the differences observed for the Mutant HTT levels among the three different brain tissues.

human-mouse-total-huntingtin-detection-kit

 

 

Validation of Total HTT assay on human and mouse neuroblastoma cell lines

Human SH-SY5Y and mouse Neuro-2a neuroblastoma cell lines were cultured in T175 flasks in complete culture medium at 37°C-5% CO2. After a 48h incubation, the cells were lysed with 3 mL of lysis buffer #2 (1X) for 30 minutes at RT under gentle shaking. After centrifugation of the cell lysates, the supernatants were serially diluted in lysis buffer #2 (1X), and 10 µL of each dilution were transferred into a low volume white microplate before the addition of 10 µL of HTRF Total HTT detection reagents.

The HTRF Total HTT assay was suitable for the measurement of the levels of soluble WT human and mouse HTT proteins present in SH-SY5Y and Neuro-2a cell lysates.

human-mouse-total-huntingtin-detection-kit

 

human-mouse-total-huntingtin-detection-kit

 

 

Resources

1-2 of 2 Resources
Guide Icon
Guide
HTRF solutions, guide to major applications

This guide provides you an overview of HTRF applications in several therapeutic areas.

Application Note Icon
Application Note
Simple and sensitive HTRF mutant and total HTT immunoassays to speed up the discovery of new drugs against Huntington’s disease

Dive deeper into Huntington’s disease research with this application note

Huntington’s disease is a neurodegenerative disorder that...

SDS, COAs, Manuals and more Illuminator

SDS, COAs, Manuals and more

Are you looking for technical documents for this product. We have housed them in a dedicated section., click on the links below to explore.

Scroll Icon