Alpha-Tubulin Housekeeping Cellular Kit HTRF®

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.

Ease-of-use
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 Cisbios 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 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.

1.Validation on adherent cell lines from different species and tissue types

Human cell lines (HEK293, HeLa, SH-SY5Y, LX-2* and IMR-90), mouse cell lines (NIH/3T3 and C2C12), and the hamster cell line CHO were plated in 96-well culture plates at different cell densities and incubated at 37°C, 5% CO2 for 24 hours. Cells were then lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking, and 4 µL of cell lysate (neat or prediluted in the supplemented lysis buffer) were transferred into a low volume white microplate. For HTRF detection, 12 µL of kit diluent were added followed by the dispensing of 4 µL of premixed detection antibodies. The HTRF signal was recorded after an overnight incubation at RT.

Alpha-Tubulin housekeeping assay on HEK293

Alpha-Tubulin housekeeping assay on HeLa

Alpha-Tubulin housekeeping assay on NIH3T3

Alpha-Tubulin housekeeping assay on SH-SY5Y

Alpha-Tubulin housekeeping assay on C2C12

Alpha-Tubulin housekeeping assay on IMR90

The HTRF Alpha-Tubulin Housekeeping kit is adapted to adherent cells from different tissue types (kidney, brain, liver, lung, muscle, ovary, cervix). It has been validated on samples from human, mouse and hamster origin, and is also compatible with rat, monkey, dog and pig species. The assay range and sensitivity have been optimized on the most commonly used cell lines, using classical cell densities to avoid cell lysate predilution before HTRF detection. For just a few cell lines that express high levels of alpha-tubulin (e.g. C2C12 and LX-2*), the lysate must be prediluted in the supplemented lysis buffer before transfer to the detection plate (dilution factor between 1/2 and 1/10 at the most).

2. Validation on suspension cell lines and primary cells

The human lymphoblast cell lines Jurkat and TK6, as well as PBMCs isolated from human blood samples, were plated in half 96-well plates at different cell densities (under 30 µL) and directly lysed with 10 µL of supplemented lysis buffer #4 (4X) for 30 minutes at RT under gentle shaking. For HTRF detection, 4 µL of neat cell lysate were transferred into a low volume white microplate and 12 µL of kit diluent were added before the dispensing of 4 µL of premixed detection antibodies. The HTRF signal was recorded after an overnight incubation at RT. The assay is also suitable for working on common cell densities of T- and -B lymphoblast cell lines and PBMCs, without the need to predilute cell lysates.

Alpha-Tubulin housekeeping assay on Jurkat

Alpha-Tubulin housekeeping assay on human PBMCs

3. Validation on tissue samples

Liver tissue lysates from three DIN (Diet-Induced NASH) mice* were prepared with lysis buffer #3 and analyzed as described in the technical note Optimize your HTRF® cell signaling assays on tissues. After total protein quantitation, samples were adjusted to the same concentration and serially diluted in the supplemented lysis buffer. For HTRF detection, 4 µL of each tissue lysate dilution were transferred into a low volume white microplate and 12 µL of kit diluent were added, before the dispensing of 4 µL of premixed detection antibodies. The HTRF signal was recorded after an overnight incubation at RT. The Alpha-Tubulin Housekeeping assay is compatible with tissue lysates, with an assay range adapted to samples containing low, medium and high concentrations of proteins. *DIN mouse liver samples were kindly provided by the preclinical CRO Physiogenex (Labège, France).

Alpha-Tubulin housekeeping assay on liver tissue lysates

4. Correlation with the BCA protein assay

Lysates of human and mouse cell lines plated at different cell densities in 96-well culture plates for 24 hours were prepared as described in Section 1. Serial dilutions of liver tissue lysates from DIN (Diet-Induced NASH) and MCD (Methionine- and -Choline Deficient) mice* were prepared as described in the previous section. For HTRF detection of the housekeeping protein alpha-tubulin, 4 µL of cell or tissue lysate were transferred into a low volume white microplate before the addition of 12 µL of kit diluent and 4 µL of premixed detection antibodies. In parallel, the concentration of proteins was determined in the same lysates using the BCA protein assay (QuantiPro BCA Assay Kit, Sigma-Aldrich) according to the manufacturers intructions. For both methods (HTRF or BCA), lysates were analyzed either neat or prediluted in the appropriate buffer in order to work within the linear range of each kit. The cell densities used for the correlations are mentioned on each graph next to each point.

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.

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Cisbio lysis buffer compatibility

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physiologically relevant results fo fast flowing research - Flyers

Species compatibility

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HTRF Alpha-tubulin Housekeeping kit

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Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalogue

A guide to Homogeneous Time Resolved Fluorescence

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Get the brochure about technology comparison. - Brochures

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Seeding and lysing recommendations for a number of cell culture vessels. - Notes techniques

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Manual a-Tubulin housek Kit / 64atubpeg-64atubpeh

64atubpeg-64atubpeh - Protocoles

Certificate of Analysis a-Tubulin housek Kit / 64ATUBPEG

64ATUBPEG Lot 10H - Rapports de contrôle qualité

Safety Data Sheet (DEU) a-Tubulin housek Kit / 64ATUBPEG