Description
Alpha-Tubulin is a member of the tubulin protein superfamily, which polymerizes with the beta tubulin to form microtubules, a major component of the eukaryotic cytoskeleton. Microtubules contribute to several cellular processes, such as structural support, intracellular transport, and DNA segregation. The Alpha-Tubulin kit is designed for the quantitative detection of total Alpha-Tubulin in cell lysates. This assay can be used as a normalization readout.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
Assay principle
Alpha-Tubulin is measured using a sandwich immunoassay involving two specific anti-alpha-Tubulin antibodies, respectively labelled with Europium Cryptate (donor) and d2 (acceptor). The intensity of the signal is proportional to the concentration of the total alpha-Tubulin present in the sample.
Assay protocol
The protocol for the Alpha-Tubulin assay is described here. Cells are plated, stimulated, and lysed in the same 96-well culture plate. Lysates are then transferred into the assay plate for the detection of alpha-Tubulin by HTRF reagents. This protocol enables the cells' viability and confluence to be monitored. The antibodies labelled with HTRF fluorophores may be pre-mixed and added in a single dispensing step to further streamline the assay procedure. The assay detection can be run in 96- to 384-well plates by simply resizing each addition volume proportionally.
Alpha tubulin 2-plate assay protocol
12,500 NIH/3T3 cells were plated in 96-well plate and treated after incubation overnight for 16h with compounds at 37°C, CO2. Lysis buffer was added after supernatant removal. 30 minutes under shaking conditions permit the lysate generation and then we transferred the lysatesd to 384-well plate for detection. Trichostatin A (TSA), a potent inhibitor of histone deacetylase, and Tubacin, a selective inhibitor of HDAC6, increase the level of acetylated α –Tubulin on Lysine 40 on NIH/3T3 cells while the expression level of α –Tubulin in presence of both inhibitors remains constant.
