AF641, AI954 and AW954 antibodies label markers of the human endoplasmic reticulum by western blot

Authors

  • Lucie Mencier
  • Lana Moudarres
  • Gaelle Najand
  • Laura Roch
  • Audrey Ruetsche
  • Valerie Schwitzguebel
  • Wissem Seddiki
  • Paola Soulie
  • Nneka Anagbogu
  • Kenza Bennani
  • Elise Brun
  • Hannah Butterworth
  • Jennifer Carry
  • Soyan Dawit
  • Meg-Mai-Ly Diep
  • Odyssee Ferrillo
  • Maxime Guertler
  • Catalina Gureu
  • Margaux Herren
  • Simon Leyss
  • Karolina Liborova
  • Marie Maître
  • Livia Manghetti
  • Stéphane Durual
  • Cyril Guilhen Cyril.Guilhen@unige.ch
  • Tania Jauslin

DOI:

https://doi.org/10.24450/journals/abrep.2023.e1227

Abstract

The recombinant antibodies AF641, AI954 and AW954 detect different markers of the endoplasmic reticulum by western blot.

Introduction

BiP (UniProt #P11021; binding immunoglobulin protein, also known as GRP78) and endoplasmin (UniProt #P14625) are molecular chaperones localized in the endoplasmic reticulum (ER). BiP and endoplasmin play critical roles in the folding of proteins in the secretory pathway (Melnick et al., 1994; Csermely et al., 1995). A KDEL sequence at the luminal C-terminal end of BiP, endoplasmin, and other proteins ensures their specific localization in the ER (Munro and Pelham., 1987). In the present study, we assessed the ability of six recombinant antibodies to detect BiP, endoplasmin and the KDEL motif in HEK293 cells by western blot. Antibodies AF641 and AI954 were able to recognize the human BiP protein. The KDEL motif was detected by the antibody AW954. The other tested antibodies failed to label either BiP (AF650 and AF637) or the human endoplasmin (AP498).

Materials & Methods

Antibodies: ABCD_AF637, ABCD_AF641, ABCD_AF650, ABCD_AI954, ABCD_AP498 and ABCD_AW954 antibodies (ABCD nomenclature, http://web.expasy.org/abcd/) were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) as mini-antibodies with the antigen-binding portion fused to a rabbit IgG Fc (see Table 1 for clone names and references). HEK293 suspension cells (growing in HEK TF medium, Xell#861-0001, supplemented with 0.1% Pluronic F68, Sigma #P1300) were transiently transfected with a vector coding for the scFv-Fc of each antibody. Supernatants (see Table 1 for individual yields) were collected after 4 days.

ABCD Clone Reference Yield (mg/L) Target
AF637 GC-20 Naoki et al., 2010 <5 BiP
AF641 B4 Pasqualini et al., 2018 20 Bip
AF650 2D6F9 Hallahan and Yan, 2017 20 BiP
AI954 MAb159 Gill and Liu, 2018 <5 BiP
AP498 H11B Arnold-Shild et al., 2000 120 Endoplasmin
AW954 VHH 5 Klooster et al., 2009 10 KDEL
Table 1. Clone numbers, references, production yields and targets of the antibodies used in this study.

Protocol: 2.5x105 HEK293 cells were pelleted, washed once in PBS and lysed in PBS containing 0.5% (v/v) Triton X-100. Nuclei were pelleted by centrifugation (5 min at 12’000 g) and the supernatant was recovered and mixed with reducing or non-reducing sample buffer (20.6% (w/v) sucrose, 100 mM Tris pH 6.8, 10 mM EDTA, 0.1% (w/v) bromophenol blue, 4% (w/v) SDS, +/- 6% (v/v) Beta-mercaptoethanol). Each sample was separated by electrophoresis on an acrylamide gel 4-20% (SurePAGE Bis-Tris, Genscript #M00655) (200 V, 30 min), and transferred onto a nitrocellulose membrane using a dry transfer system for 10 min (iBlot gel transfer device, Invitrogen #IB1001EU). The membranes were blocked 2 h in PBS containing 0.1% (v/v) Tween20 and 7% (w/v) milk and washed three times for 5 min in PBS + 0.1% (v/v) Tween20. The membranes were then incubated with the recombinant antibodies (5 mg/L in PBS-Tween-milk) for 20 min at room temperature and washed three times for 5 min. The membranes were then incubated for 20 min with horseradish peroxidase-coupled goat anti-rabbit IgG (Sigma-Aldrich #A8275, dilution 1:3000 in PBS-Tween-milk) and washed 5 times for 5 min in PBS-Tween. The signal was revealed by enhanced chemiluminescence (ECL) (Millipore) using a PXi-4 gel imaging system (Syngene).

Results

Both antibodies AF641 and AI954 recognize a similar single band in reducing and non-reducing conditions at approximately 80 kDa. The observed signal is slightly higher than the expected theoretical size of the human BiP protein (72 kDa) and may be due to known post-translational modifications of the BiP protein. No signal was detected with others anti-BiP antibodies (AF637 and AF650, Fig. 1). The absence of signal with AF637 might be due to the fact that this antibody is poorly produced. The antibody AW954 recognizes several proteins which are presumed to bear a C-terminal KDEL retention motif (Fig. 1). One of the proteins detected by AW954 migrates with an apparent size of 80 kDa and may correspond to the BiP protein. No signal was observed with the AP498 anti-endoplasmin antibody.

Figure 1. AF641, AI954 and AW954 specifically label markers of endoplasmic reticulum in HEK293 cells in both reducing (R) and non-reducing (NR) conditions. No signal was observed with AF637, AF650 and AP498 antibodies.

Conflict of interest

Tania Jauslin is an associate-editor of the journal Antibody Reports.

References

Arnold-Schild D, Kleist C, Welschof M, Opelz G, Rammensee HG, Schild H, et al. One-Step Single-Chain Fv Recombinant Antibody-based Purification of gp96 for Vaccine Development. Cancer Research. 2000; 60(15): 4175‑8. PMID: 10945626.

Csermely P, Miyata Y, Schnaider T, Yahara I. Autophosphorylation of grp94 (Endoplasmin). Journal of Biological Chemistry. 1995; 270(11): 6381‑8. PMID: 7890776.

Gill P, Liu R. Antibodies that bind cell surface GRP78 and their use for detection of cancer. Unites States, US20180094054. 2018.

Hallahan D, Yan H. Antibodies to GRP78. Unites States, US20170298142. 2017.

Klooster R, Eman MR, le Duc Q, Verheesen P, Verrips CT, Roovers RC, et al. Selection and characterization of KDEL-specific VHH antibody fragments and their application in the study of ER resident protein expression. Journal of Immunological Methods. 2009; 342(1): 1‑12. PMID: 19041652.

Melnick J, Dul JL, Argon Y. Sequential interaction of the chaperones BiP and GRP94 with immunoglobulin chains in the endoplasmic reticulum. Nature. 1994; 370(6488): 373‑5. PMID: 7913987.

Munro S, Pelham HRB. A C-terminal signal prevents secretion of luminal ER proteins. Cell. 1987; 48(5): 899‑907. PMID: 3545499.

Naoki K. Pharmaceutical composition comprising anti-GRP78 antibody as active ingredient. Unites States, US20100041074. 2010.

Pasqualini R, Arap W, Staquicini F, Ferrara F, D'Angelo S, Bradbury A. EPHA5 and GRP78 binding antibodies and uses thereof and selection of phage-displayed accessible recombinant targeted antibodies. World. WO2018057703. 2018.

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Published

2023-07-18

Section

Article

How to Cite

1.
Mencier L, Moudarres L, Najand G, Roch L, Ruetsche A, Schwitzguebel V, Seddiki W, Soulie P, Anagbogu N, Bennani K, Brun E, Butterworth H, Carry J, Dawit S, Diep M-M-L, Ferrillo O, Guertler M, Gureu C, Herren M, Leyss S, Liborova K, Maître M, Manghetti L, Durual S, Guilhen C, Jauslin T. AF641, AI954 and AW954 antibodies label markers of the human endoplasmic reticulum by western blot. Antib. Rep. [Internet]. 2023 Jul. 18 [cited 2024 May 22];6(1):e1227. Available from: https://oap.unige.ch/journals/abrep/article/view/1227

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