Alpha-Tubulin Housekeeping Cellular Kit

This assay enables measurement of the endogenous expression level of alpha-tubulin in cells and tissues to normalize data obtained on the protein(s) of interest.
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  • Ease-of-use Ease-of-use
  • High sensitivity High sensitivity
This assay enables measurement of the endogenous expression level of alpha-tubulin in cells and tissues to normalize data obtained on the protein(s) of interest.
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Overview

This assay is designed to measure the endogenous expression level of alpha-tubulin in cells and tissues in order to normalize data obtained on the phospho- and/or total protein(s) of interest. Due to its essential role in the cytoskeletons of cells, alpha-tubulin is ubiquitously and constitutively expressed in every tissue. Moreover, its amino acid sequence is highly conserved and is thus identical in almost all species. These characteristics make it one of the most commonly used housekeeping proteins. Its concentration is proportional to cell numbers and total protein concentrations, and it is therefore used as an internal control for data normalization to correct for signal changes caused by experimental variability (e.g. number of cells remaining in the culture plate after treatment, or lysis efficacy). The low sample volume of 4 µL and its compatibility with all Cisbio’s lysis buffers enable a multi-parametric analysis with the detection of alpha-tubulin in parallel with the phospho- and/or total protein(s) on the same lysate.

Benefits

  • DATA NORMALIZATION

Alpha-Tubulin Housekeeping assay principle

The Alpha-Tubulin Housekeeping assay measures endogenous alpha-tubulin in a cell or tissue lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Alpha-Tubulin Housekeeping assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of alpha-tubulin in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing any changes caused by experimental variability under a no-wash assay format.
Alpha-Tubulin Housekeeping assay principle

Alpha-Tubulin Housekeeping 2-plate assay protocol

The assay is run under a two-plate assay protocol, where cells are plated and treated in a culture plate. For detection, 4 µL of lysate are subsequently transferred into a low volume 96- or 384-well white plate, and 12 µL of kit diluent are added before dispensing 4 µL of HTRF® reagents. This protocol enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.
Alpha-Tubulin Housekeeping 2-plate assay protocol

Alpha-tubulin simplified pathway

Alpha-tubulin forms heterodimers with beta-tubulin, which are the building blocks incorporated into protofilaments, and then cylindric structures called microtubules. Microtubules are found throughout the cytoplasm of all eukaryotic cells and are key elements of the cytoskeleton with crucial functions in cell shape, organization, movement, and division. Due to its essential role, the alpha-tubulin protein is ubiquitously and constitutively expressed in every tissue. Moreover, its amino acid sequence is highly conserved in evolution and is thus identical in almost all species. These characteristics make it one of the most commonly used housekeeping proteins.
Alpha-tubulin signaling pathway
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