AF397, AK652, AN193 and AV442 antibodies recognize a GFP-tagged protein by western blot

Authors

  • Anna Marchetti

DOI:

https://doi.org/10.24450/journals/abrep.2022.e822

Abstract

AF397, AK652, AN193 and AV442 antibodies against the GFP protein recognize a GFP-tagged human TAC protein by western blot.

Introduction

The green fluorescent protein (GFP) (Uniprot P42212) is a protein tag originally isolated from the jellyfish Aequorea victoria, widely used as a fluorescent reporter to detect and visualize GFP-fused proteins (Tsien, 1998). The AF397, AK652, AN193 and AV442 recombinant antibodies detect a GFP-tagged human TAC/IL2RA protein by western blot.

Materials & Methods

Antibodies: The anti-GFP ABCD_AF397, ABCD_AK652, ABCD_AN193 and ABCD_AV442 antibodies (http://web.expasy.org/abcd/, ABCD nomenclature) were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) as mini-antibodies with the antigen-binding domain fused to a rabbit IgG Fc. The synthesized antibody sequences (GeneArt, Invitrogen) correspond to the sequences of the variable regions joined by a peptide linker (GGGGS)3 (Table 1). HEK293 suspension cells (growing in HEK TF medium, Xell 861-0001, supplemented with 0.1% Pluronic F68, Sigma P1300) were transiently transfected with the vector coding for the mini-antibodies. Supernatants (see Table 1 for individual yields) were collected after 4 days. The recombinant antibody AJ519, which recognizes human TAC/IL2RA protein (Uniprot P01589), was used as a positive control (Arsimoles et al., 2020).

ABCD Clone Binder type Reference Yield (mg/L)
AF397 LaG-2 VHH Fridy et al., 2014 140
AK 652 BH-GBP2 VHH Pellis et al., 2012 120
AN193 3G86.32 DARPin Brauchle et al., 2014 50
AV442 N86/44.1 scFv Andrews et al., 2019 120
AJ519 7G7 scFv Arsimoles et al., 2020 40
Table 1. Clone number, epitope, reference and production yields for the antibodies used in this study.

Antigen: HEK cells (growing in DMEM GlutaMAXTM, Gibco 31966; supplemented with 8% Fetal Bovine Serum, Gibco 10270), transiently transfected 2 days before the experiment with a C-terminally GFP-tagged human TAC/IL2RA protein (Uniprot P01589), were used to detect the protein tag.

Protocol: 5x106 cells were pelleted and lysed for 15 min in 100 L of ice-cold lysis buffer (25 mM Tris-HCl pH 7.4 + 0.5 % Triton X-100 + 120 mM NaCl) containing protease inhibitors. Lysate was centrifuged 15 min, 10’000 g at 4 °C to remove nuclei. One volume of sample buffer was added to the lysate (20.6% (w/v) sucrose, 100 mM Tris pH 6.8, 10 mM EDTA, 0.1% (w/v) bromophenol blue, 4% (w/v) SDS). Dilutions of each sample (500’000 and 100’000 cells) were migrated (150 V, 45 min) in a 4-20% acrylamide gel (Genscript, SurePAGE Bis-Tris, M00655), and transferred to a nitrocellulose membrane using a dry transfer system for 7 minutes (iBlot gel transfer device, Invitrogen IB23001). The membranes were blocked during 60 min in PBS containing 0.1% (v/v) Tween20 and 7% (w/v) milk, and washed once for 15 minutes in PBS + 0.1% (v/v) Tween20 (PBS-Tween). The membranes were then incubated overnight at RT with the primary antibodies (final concentration 5 mg/L in PBS-Tween). The membranes were then washed three times (15+15+10 min) in PBS-Tween, incubated for 1 hour with the horseradish peroxidase-coupled goat anti-rabbit IgG (Sigma-Aldrich A8275, dilution 1:3000) and washed three times (15 min) in PBS-Tween. The signal was revealed by enhanced chemiluminescence (ECL) (Amersham Biosciences) using a PXi-4 gel imaging systems (Syngene).

Results

AF397, AK652, AN193 and AV442 antibodies specifically recognized GFP in cells transfected with the GFP-tagged TAC/IL2RA protein, detecting two bands between 60 and 70 kDa (corresponding to immature and mature glycosylation forms, respectively; Fig. 1). The same bands were seen with AJ519, an anti-TAC recombinant antibody used as positive control (Fig. 1). In addition, anti-GFP antibodies detected a smaller protein (37 kDa) which presumably corresponds to a proteolytically processed form of the IL2RA-GFP protein (Fig. 1). The specificity of the signal was verified by the absence of anti-GFP staining in non-transfected cells (Fig. 1, “NT”).

Figure 1. AF397, AK652, AN193 and AV442 specifically recognize GFP in HEK cells expressing a GFP-tagged TAC/IL2RA protein (“+TAC-GFP”). Two bands, indicated by arrowheads (shown only for AF397), correspond to different glycosylation states of the IL2RA protein. The same bands are seen for the positive control, labeled with the anti-TAC AJ519 antibody (“AJ519-Ctr”). The anti-GFP antibodies also detect free GFP (around 35 kDa, asterisk). No labelling was seen in non-transfected cells (“NT”). For each antibody and condition, two sample dilutions were used: 500’000 and 100’000 cells/well (labels shown only for AF397).

Conflict of interest

The authors declare no conflict of interest.

References

Andrews NP, Boeckman JX, Manning CF, et al. A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain. Elife. 2019; 8:e43322. PMID: 30667360

Arsimoles D, D’Esposito AG, Gaspoz V, et al. The AJ519 antibody labels the human TAC/IL2RA protein by immunofluorescence. Antibody Reports, 2020, 3:e118. doi:10.22450/journals/abrep.2020.e118

Brauchle M, Hansen S, Caussinus E, et al. Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry. Biol Open. 2014; 3(12):1252-61. PMID: 25416061

Fridy PC, Li Y, Keegan S, et al. A robust pipeline for rapid production of versatile nanobody repertoires. Nat Methods. 2014; 11(12):1253-60. PMID: 25362362

Pellis M, Pardon E, Zolghadr K, et al. A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional nanobodies. Arch Biochem Biophys. 2012; 526(2):114-23. PMID: 22583807

Tsien RY. The green fluorescent protein. Annu Rev Biochem. 1998; 67:509-44. PMID:9759496

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Published

2022-05-17

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Article

How to Cite

1.
Marchetti A. AF397, AK652, AN193 and AV442 antibodies recognize a GFP-tagged protein by western blot. Antib. Rep. [Internet]. 2022 May 17 [cited 2024 May 21];5(1):e822. Available from: https://oap.unige.ch/journals/abrep/article/view/822

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