Alpha-Tubulin Housekeeping Cellular Kit

This assay is designed to measure the endogenous expression level of alpha-tubulin in cells and tissues to normalize data obtained on the phospho- and/or total protein(s) of interest.

Due to its essential role in the cytoskeleton 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 number and total protein concentration, 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 the compatibility with Cisbio’s lysis buffers #1, #2, #3 and #4 enables a multi-parametric analysis with the detection of alpha-tubulin in parallel with the phospho- and/or total protein(s) on the same lysate.

Download Alpha-tubulin housekeeping assay application notes

Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s Alpha-Tubulin Housekeeping assay is based on a TR-FRET sandwich immunoassay format comprising two specific anti-alpha-tubulin antibodies, one labeled with Eu3+cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind to alpha-tubulin, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a low volume 96- or 384-well plate format, but can easily be further miniaturized or upscaled. Only 4 µL of sample is needed, enabling the detection of the phospho- and/or total protein of interest in parallel on the same lysate. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assay can be run with frozen cell or tissue lysates, or with fresh cells in culture. After lysis, endogenous alpha-tubulin can be quantitatively detected using the HTRF Alpha-Tubulin Housekeeping cellular assay kit reagents and most TR-FRET multimode plate readers.

Image of the HTRF assay principle for cellular alpha-tubulin housekeeping protein detection

A simpler, more flexible assay protocol - adapted to your applications

Two-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 two-plate assay protocol

Product performances

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. 

Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping assay on adherent cell lines
Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping assay on adherent cell lines
Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping assay on adherent cell lines
Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping assay on adherent cell lines
Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping assay on adherent cell lines
Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping assay on adherent cell lines
Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping assay on adherent cell lines
Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping assay on adherent cell lines

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.

Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping assay on suspension cells
Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping assay on suspension cells
Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping assay on suspension cells

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.

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.

Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping on mouse liver tissue samples

*DIN mouse liver samples were kindly provided by the preclinical CRO Physiogenex (Labège, France).

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 manufacturer’s 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.

Correlation on human and mouse cell lines

Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping assay on adherent cell lines
Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping assay on adherent cell lines
Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping assay on adherent cell lines
Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping assay on adherent cell lines

Correlation on tissue samples

Image of the validation of the Cisbio HTRF Alpha-Tubulin Housekeeping assay on adherent cell lines

*DIN and MCD mouse liver samples were kindly provided by the preclinical CRO Physiogenex (Labège, France).

Using human cell lines, mouse cell lines, or tissues, the level of alpha-tubulin measured by HTRF is always correctly correlated to the concentration of proteins determined by the BCA method (with R2 > 0.91). These results demonstrate that alpha-tubulin is an appropriate housekeeping protein suitable for data normalization.

5. Comparison with Western blot

The human hepatoma cell line HepG2 was plated in a 96-well culture plate at different cell densities (from 3,125 to 25,000 cells/well) 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. 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 followed by the dispensing of 4 µL of premixed detection antibodies. The HTRF signal was recorded after an overnight incubation at RT. Equal amounts of the same lysates were used for a side by side comparison with Western Blot.

Using the lowest cell density (3,125 cells plated/well), the Western Blot method is not sensitive enough to detect a significant signal in the cell lysate. Conversely, the HTRF® Alpha-Tubulin Housekeeping assay enables a comfortable signal detection with a signal over negative (S/N) of 5. The HTRF® Alpha-Tubulin Housekeeping assay is therefore more sensitive than the Western Blot.

graph of comparison-western blot hepg2 cell lysates

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.

Image of alpha tubulin simplified pathway

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Alpha-Tubulin Housekeeping kit - 500 tests64ATUBPEG
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Alpha-Tubulin Housekeeping kit - 10,000 tests64ATUBPEH
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Alpha-Tubulin Housekeeping kit - 50,000 tests64ATUBPEY
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Companion products

DescriptionCat. noProduct insertMSDS
Alpha-Tubulin Housekeeping kit control lysate64ATUBTDA
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Documents