The AA345 antibody specifically recognizes α-tubulin by western blot in human cell lines

Authors

  • Lucie Oberhauser
  • Miriam Stoeber

DOI:

https://doi.org/10.24450/journals/abrep.2020.e260

Abstract

The recombinant antibody AA345 specifically detects human α-tubulin in HEK293, Hela, and HepG2 human cell lines by western blot.

Introduction

The recombinant AA345 antibody was previously shown to successfully recognize the human α-tubulin by immunofluorescence (Guerreiro and Meraldi, 2019; Lima and Cosson, 2019). Here, we add a new application to the AA345 antibody by describing its ability to detect the human α-tubulin in cell lysates by western blot.

Materials & Methods

Antibodies: ABCD_AA345 antibody (ABCD nomenclature, http://web.expasy.org/abcd/) was produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) as a mini-antibody with the antigen-binding scFv fused to a mouse IgG2a Fc. The synthesized scFv sequence (GeneArt, Invitrogen) corresponds to the sequences of the variable regions of the clone F2C (Nizak et al., 2003) joined by a peptide linker (GGGGS)3. HEK293 suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco #12338) were transiently transfected with the vector coding for the scFv-Fc. Supernatants (~100 mg/L) were collected after 4 days.

Cell culture and lysis: HEK293 and Hela cells were grown in high glucose DMEM without pyruvate (Gibco, #41965-039) supplemented with 10% FBS. HepG2 cells were grown in low glucose DMEM with pyruvate (Gibco, #31885-023) supplemented with 10% FBS. Cells were seeded in 6-well plates and cultured to reach 70-80% confluency. Total cellular proteins were extracted by lysing cells in 200 μl RIPA buffer (10 mM Tris pH 7.4, 150 mM NaCl, 0.1% SDS, 1X Triton, 2 mM EDTA). Lysates were sonicated and proteins were quantified using Pierce™ BCA protein assay kit (Thermo Scientific, #23227).

Protein separation and transfer:Cell lysates containing 20 μg of total protein were mixed with 4X NuPAGE® LDS sample buffer (Thermo Fisher Scientific, #NP0007) and 0.1 M DTT. Samples were heated at 70 °C for 10 min prior to loading in duplicate onto a Bolt™ 4-12% Bis-Tris pre-casted gel (Thermo Fisher Scientific, #NW04120BOX) and run at 150 V for 1 h along with Spectra™ Multicolor Broad Range Protein ladder (Thermo Scientific, #26634). In the meantime, a 0.45 µm PVDF membrane (Millipore, #IPVH85R) was activated in Isopropanol for 30 s and further soaked in transfer buffer (25 mM Tris, 186 mM Glycine, 20% v/v Isopropanol) along with blot paper (Bio-Rad, #1703966). After complete protein migration, the gel was equilibrated in transfer buffer for 5-10 min. Protein transfer was performed using a Trans-Blot® SD semi-dry transfer cell (Bio-Rad, #1703848) at 300 mA for 15 min.

Immunoblotting:The membrane was cut in half. Both pieces were rinsed with water and blocked with 5% BSA in TBS (Tris 20 mM, NaCl 150 mM, pH 7.6) for 1 h. Membranes were washed 2 times for 10 min in TBS-Tween 0.1% and incubated overnight at 4 °C with anti-α-tubulin primary antibodies AA345 or T5168 (Merck; Piperno et al., 1987) diluted to 1:5000 and 1:7500, respectively, in TBS-Tween 0.1% containing 5% BSA. On the next day, membranes were washed 3 times for 10 min with TBS-Tween 0.1% and incubated with HRP-conjugated anti-mouse secondary antibody (Merck, #A5278) for 1 h at RT.

Protein detection:Membranes were washed 3 times for 5 min with TBS-Tween 0.1% and antibody-bound protein bands were visualized by the addition of SuperSignal™ West Pico PLUS chemiluminescent substrate (Thermo Scientific, #34580) directly on the membranes placed in the iBRIGHT Western Blot imaging system (Thermo Fisher Scientific, #FL1500). Membranes were exposed for 10 s, 30 s, and 1 min until signal saturation.

Results

The recombinant antibody AA345 successfully and specifically detected human α-tubulin (50 kDa) in each of the tested cell lines (Fig. 1). It did so with a specificity superior to the commercial monoclonal anti-α-tubulin used as a control (T5168), which shows lower unspecific bands in both HEK293 and HepG2 cells.

Figure 1. Specific detection of human α-tubulin by western blot (predicted molecular weight: 50 kDa in reducing conditions) in lysates of HEK293, HeLa, and HepG2 cells using antibodies AA345 (left) and control T5168 (right). Detection of chemiluminescence signal upon 30 s (AA345, left) or 10 s (T5168, right) of exposure.

Conflict of interest

The authors declare no conflict of interest.

References

Guerreiro A, Meraldi P. AA344 and AA345 antibodies recognize the microtubule network in human cells by immunofluorescence. Antib. Rep. 2019; 2:e17. doi: 10.24450/ journals/abrep.2019.e17

Lima W, Cosson P. The AA344 and AA345 antibodies detect human tubulin by immunofluorescence in HeLa cells. Antib. Rep. 2019; 2:e108. doi: 10.24450/ journals/abrep.2019.e108

Nizak C, Martin-Lluesma S, Moutel S, Roux A, Kreis TE, Goud B, et al. Recombinant antibodies against subcellular fractions used to track endogenous Golgi protein dynamics in vivo. Traffic. 2003; 4(11):739-53. PMID: 14617357

Piperno G, LeDizet M, Chang XJ. Microtubules containing acetylated alpha-tubulin in mammalian cells in culture. J Cell Biol. 1987; 104(2):289-302. PMID: 2879846

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Published

2020-11-17

Section

Article

How to Cite

1.
Oberhauser L, Stoeber M. The AA345 antibody specifically recognizes α-tubulin by western blot in human cell lines. Antib. Rep. [Internet]. 2020 Nov. 17 [cited 2024 May 27];3(4):e260. Available from: https://oap.unige.ch/journals/abrep/article/view/260