Erythropoietin/EPO Antibody (AE7A5) [Unconjugated]

Analysis of the expression of EPO-IgG1 variants. Total soluble protein (TSP) extracts from N. benthamiana delta XTFT leaves expressing different EPO-Fc variants were analyzed by immunoblotting at 5 days post-infiltration (dpi) using (A) anti-EPO and (B) anti-hIgG antibodies. (C) EPO-Fc variants purified out of TSP with protein A were stained with Coomassie Brilliant Blue under reducing conditions (CBB). (D) Oligomerization of EPO-Fc variants was evaluated in 8% native PAGE of TSP non-reducing samples. Ponceau staining shows similar amounts of protein loaded. Protein size markers are shown in kilo Dalton (kDa). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/34305971), licensed under a CC-BY license. Not internally tested by R&D Systems.

Western blots of mock-treated and alpha -L-arabinofuranosidase-digested samples from rhEPO-producing Physcomitrella lines. Ten microgram total protein of precipitated culture supernatants of the rhEPO-producing lines 174.16, delta galt1 and X24 were digested with one unit of alpha -L-arabinofuranosidase ( alpha -Arafase), while control samples were treated equivalently but without alpha -L-arabinofuranosidase (mock). After separation on SDS-PAGE and blotting, the PVDF-membrane was subsequently incubated with the anti-1,5-alpha -L-arabinan antibody (LM6-M, 1:10) (A) and an anti-hEPO monoclonal antibody (1:4,000) (B). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35252146), licensed under a CC-BY license. Not internally tested by R&D Systems.

Western blots of precipitated culture supernatants of rhEPO-producing Physcomitrella lines. Five microgram total protein of the precipitated and blotted culture supernatants of the rhEPO-producing lines 174.16, delta galt1 and X24 were subsequently immunodetected with the anti-1,5-alpha -L-arabinan antibody (LM6-M, 1:10) (A) and the anti-hEPO monoclonal antibody (1:4,000) (B). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35252146), licensed under a CC-BY license. Not internally tested by R&D Systems.

STABILON significantly boosts the intracellular and secreted levels of hEPO in mammalian cells. (a) Stably transfected CHO-K1 cells expressing hEPO (lane 2), hEPO-Stab (lane 3) or hEPO-2 × Stab (lane 4) proteins were induced by doxycycline treatment for 24 h. Lane 1 is the control nontransfected cell. Total cell extracts were fractionated on SDS-PAGE. Expression of hEPO derivatives (unmodified and glycosylated forms, Form I–III) was analyzed by immunoblotting using anti-hEPO monoclonal antibody. Coomassie-stained gel shows the protein loading (left panel). (b) 10 µL of cell culture medium of doxycycline-induced control (lane 1), hEPO (lane 2) or hEPO-Stab (lane 3) expressing stably transfected CHO-K1 cells were fractionated on SDS-PAGE. Secreted hEPO glycosylated species (Form III) were analyzed by immunoblotting. Coomassie-stained gel serves as loading control (left panel). Asterisk labels the extremely abundant serum albumin present in the medium. (c) Stable transfected CHO-K1 cells expressing hEPO, hEPO-Stab or hEPO-2 × Stab were induced with doxycycline treatment for 5 h, then continued for an additional 5 h in the presence of 0 μM (lanes 2, 5 and 8), 5 μM (lanes 3, 6 and 9) and 10 μM (lanes 4, 7 and 10) of MG132. Accumulation of hEPO derivatives was analyzed in total cell extracts by immunoblotting. Protein extracts of CHO-K1 cells served as control (lane 1). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35897744), licensed under a CC-BY license. Not internally tested by R&D Systems.

STABILON significantly boosts the intracellular and secreted levels of hEPO in mammalian cells. (a) Stably transfected CHO-K1 cells expressing hEPO (lane 2), hEPO-Stab (lane 3) or hEPO-2 × Stab (lane 4) proteins were induced by doxycycline treatment for 24 h. Lane 1 is the control nontransfected cell. Total cell extracts were fractionated on SDS-PAGE. Expression of hEPO derivatives (unmodified and glycosylated forms, Form I–III) was analyzed by immunoblotting using anti-hEPO monoclonal antibody. Coomassie-stained gel shows the protein loading (left panel). (b) 10 µL of cell culture medium of doxycycline-induced control (lane 1), hEPO (lane 2) or hEPO-Stab (lane 3) expressing stably transfected CHO-K1 cells were fractionated on SDS-PAGE. Secreted hEPO glycosylated species (Form III) were analyzed by immunoblotting. Coomassie-stained gel serves as loading control (left panel). Asterisk labels the extremely abundant serum albumin present in the medium. (c) Stable transfected CHO-K1 cells expressing hEPO, hEPO-Stab or hEPO-2 × Stab were induced with doxycycline treatment for 5 h, then continued for an additional 5 h in the presence of 0 μM (lanes 2, 5 and 8), 5 μM (lanes 3, 6 and 9) and 10 μM (lanes 4, 7 and 10) of MG132. Accumulation of hEPO derivatives was analyzed in total cell extracts by immunoblotting. Protein extracts of CHO-K1 cells served as control (lane 1). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35897744), licensed under a CC-BY license. Not internally tested by R&D Systems.

The effect of STABILON derivatives on the accumulation of hEPO in mammalian cells. (a) hEPO (lane 2), hEPO-StabDm1−15 (lane 3), hEPO-StabDm1−13 (lane 4), hEPO-StabDm1−10 (lane 5), hEPO-StabDm1−9 (lane 6), hEPO-StabDm1−8 (lane 7), hEPO-StabDm8−10 (lane 8), hEPO-StabDm8−11 (lane 9), hEPO-StabDm8−12 (lane 10), hEPO-StabDm1−7 (lane 11), hEPO-StabDm1−5 (lane 12), hEPO-StabDm1−3 (lane 13), hEPO-StabDm1−13-KR (lane 14), hEPO-StabDm1−13-KA (lane 15) and hEPO-StabDm1−13-DA (lane 16) transgenes in stably transfected CHO-K1 cells were induced by doxycycline-treatment for 24 h, respectively. The intracellular accumulation of hEPO derivatives was analyzed in total cell extracts by Western blotting using anti-hEPO monoclonal antibody. alpha Tubulin served as loading control. The lower diagram shows the amino acid positions and composition of the STABILON derivatives fused to hEPO. (b) hEPO (lane 2), hEPO-StabDm1-13 (lane 3) or hEPO-StabHs1–13 (lane 4) were expressed in stably transfected CHO-K1 cells by doxycycline-treatment for 24 h. Accumulation of hEPO derivatives was analyzed in total cell extracts by Western blotting using anti-hEPO monoclonal antibody. Protein extracts of CHO-K1 cells serve as control (lane 1). (c) Stably transfected CHO-K1 cells carrying the hEPO (lane 2), hEPO-StabDm1−13 (lane 3) and hEPO-StabHs1−13 (lane 4) genes were induced by doxycycline for 24 h. hEPO derivatives secreted into the tissue culture medium were analyzed by Western blotting with anti-hEPO monoclonal antibody. Tissue culture medium of CHO-K1 cells served as control (lane 1). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35897744), licensed under a CC-BY license. Not internally tested by R&D Systems.

The effect of STABILON derivatives on the accumulation of hEPO in mammalian cells. (a) hEPO (lane 2), hEPO-StabDm1−15 (lane 3), hEPO-StabDm1−13 (lane 4), hEPO-StabDm1−10 (lane 5), hEPO-StabDm1−9 (lane 6), hEPO-StabDm1−8 (lane 7), hEPO-StabDm8−10 (lane 8), hEPO-StabDm8−11 (lane 9), hEPO-StabDm8−12 (lane 10), hEPO-StabDm1−7 (lane 11), hEPO-StabDm1−5 (lane 12), hEPO-StabDm1−3 (lane 13), hEPO-StabDm1−13-KR (lane 14), hEPO-StabDm1−13-KA (lane 15) and hEPO-StabDm1−13-DA (lane 16) transgenes in stably transfected CHO-K1 cells were induced by doxycycline-treatment for 24 h, respectively. The intracellular accumulation of hEPO derivatives was analyzed in total cell extracts by Western blotting using anti-hEPO monoclonal antibody. alpha Tubulin served as loading control. The lower diagram shows the amino acid positions and composition of the STABILON derivatives fused to hEPO. (b) hEPO (lane 2), hEPO-StabDm1-13 (lane 3) or hEPO-StabHs1–13 (lane 4) were expressed in stably transfected CHO-K1 cells by doxycycline-treatment for 24 h. Accumulation of hEPO derivatives was analyzed in total cell extracts by Western blotting using anti-hEPO monoclonal antibody. Protein extracts of CHO-K1 cells serve as control (lane 1). (c) Stably transfected CHO-K1 cells carrying the hEPO (lane 2), hEPO-StabDm1−13 (lane 3) and hEPO-StabHs1−13 (lane 4) genes were induced by doxycycline for 24 h. hEPO derivatives secreted into the tissue culture medium were analyzed by Western blotting with anti-hEPO monoclonal antibody. Tissue culture medium of CHO-K1 cells served as control (lane 1). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/35897744), licensed under a CC-BY license. Not internally tested by R&D Systems.

• Reactivity: Hu
• Applications: WB, AP
• Clone: AE7A5
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated

Erythropoietin/EPO Antibody (AE7A5) [Unconjugated] Summary

• Immunogen: Erythropoietin/EPO synthetic peptide analog
  Ala28-Thr53
  Accession # P01588
• Specificity: Recognizes an epitope within the first 26 amino acids at the NH₂ terminus of human urinary and human recombinant EPO. Binds human and rat EPO but does not bind sheep, baboon, or dog EPO (1, 2).
• Source: N/A
• Isotype: IgG2a
• Clonality: Monoclonal
• Host: Mouse
• Gene: EPO
• Purity Statement: Protein A or G purified from hybridoma culture supernatant

Applications/Dilutions

• Dilutions:
  • Immunoaffinity Purification
  • Western Blot 1 ug/mL

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C, as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after opening.
  • 6 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in PBS. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Erythropoietin/EPO Antibody (AE7A5) [Unconjugated]

• ECYT5
• EP
• EPO
• epoetin
• Erythropoietin
• MGC138142
• MVCD2

Background

Erythropoietin (EPO) a glycoprotein produced primarily by the kidney, is the principal factor that regulates erythropoiesis by stimulating the proliferation and differentiation of erythroid progenitor cells.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for Erythropoietin/EPO Antibody (MAB2871)(20)

Publications using MAB2871

• Charli E. Harlow, Josan Gandawijaya, Rosemary A. Bamford, Emily-Rose Martin, Andrew R. Wood, Peter J. van der Most et al. Identification and single-base gene-editing functional validation of a cis-EPO variant as a genetic predictor for EPO-increasing therapies The American Journal of Human Genetics 2022-09-01 [PMID: 36055212]
• Z Rethi-Nagy, E Abraham, K Udvardy, E Klement, Z Darula, M Pal, RL Katona, V Tubak, T Pali, Z Kota, R Sinka, A Udvardy, Z Lipinszki STABILON, a Novel Sequence Motif That Enhances the Expression and Accumulation of Intracellular and Secreted Proteins International Journal of Molecular Sciences, 2022-07-25;23(15):. 2022-07-25 [PMID: 35897744] (Western Blot, Chinese Hamster, Drosophila)
• Lennard L. Bohlender, Juliana Parsons, Sebastian N. W. Hoernstein, Nina Bangert, Fernando Rodríguez-Jahnke, Ralf Reski et al. Unexpected Arabinosylation after Humanization of Plant Protein N-Glycosylation Frontiers in Bioengineering and Biotechnology 2022-02-18 [PMID: 35252146]
• Pia Gattinger, Shiva Izadi, Clemens Grünwald-Gruber, Somanath Kallolimath, Alexandra Castilho The Instability of Dimeric Fc-Fusions Expressed in Plants Can Be Solved by Monomeric Fc Technology Frontiers in Plant Science 2021-07-09 [PMID: 34305971]
• T Susantad, M Fuangthong, K Tharakaram, P Tit-Oon, M Ruchirawat, R Sasisekhar Modified recombinant human erythropoietin with potentially reduced immunogenicity Scientific Reports, 2021-01-15;11(1):1491. 2021-01-15 [PMID: 33452310] (ELISA Capture, Human)
• MJ Leiva-Carr, S Jiménez-Ch, DJ Harvey, NC Parra, KC Tavares, F Camacho, A González, O Sánchez, R Montesino, JR Toledo In vivo modification of the goat mammary gland glycosylation pathway N Biotechnol, 2020-11-04;61(0):11-21. 2020-11-04 [PMID: 33157282] (Western Blot, Human)
• Y Yasuoka, T Fukuyama, Y Izumi, Y Nakayama, H Inoue, K Yanagita, T Oshima, T Yamazaki, T Uematsu, N Kobayashi, Y Shimada, Y Nagaba, M Mukoyama, T Yamashita, Y Sato, JM Sands, K Kawahara, H Nonoguchi Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation Physiol Rep, 2020-06-01;8(12):e14485. 2020-06-01 [PMID: 32592328] (Western Blot, Human)
• J Tani, Y Ito, S Tatemichi, M Yamakami, T Fukui, Y Hatano, S Kakimoto, A Kotani, A Sugimura, K Mihara, R Yamamoto, N Tanaka, K Minami, K Takahashi, T Hirato Physicochemical and biological evaluation of JR-131 as a biosimilar to a long-acting erythropoiesis-stimulating agent darbepoetin alfa PLoS ONE, 2020-04-17;15(4):e0231830. 2020-04-17 [PMID: 32302352] (Western Blot, Human)
• CL Chin, JB Goh, H Srinivasan, KI Liu, A Gowher, R Shanmugam, HL Lim, M Choo, WQ Tang, AH Tan, T Nguyen-Khu, MH Tan, SK Ng A human expression system based on HEK293 for the stable production of recombinant erythropoietin Sci Rep, 2019-11-14;9(1):16768. 2019-11-14 [PMID: 31727983] (Western Blot, Human)
• MS Kwon, BC Koo, D Kim, YH Nam, XS Cui, NH Kim, T Kim Generation of transgenic chickens expressing the human erythropoietin (hEPO) gene in an oviduct-specific manner: Production of transgenic chicken eggs containing human erythropoietin in egg whites PLoS ONE, 2018-05-30;13(5):e0194721. 2018-05-30 [PMID: 29847554] (Western Blot, Chicken)
• Barbora Fecková, Patrícia Kimáková, Lenka Ilkovičová, Erika Szentpéteriová, Nataša Debeljak, Zuzana Solárová et al. Far-western blotting as a solution to the non-specificity of the anti-erythropoietin receptor antibody Oncology Letters 2016-08-01 [PMID: 27446474]
• de Lucas Cerrillo A, Bond W, Rex T Safety and angiogenic effects of systemic gene delivery of a modified erythropoietin. Gene Ther, 2015-02-26;22(5):365-73. 2015-02-26 [PMID: 25716531] (Functional Assay, Hamster)
• Park S, Won S, Song J, Kambe T, Nagao M, Kim W, Moon S EPO gene expression promotes proliferation, migration and invasion via the p38MAPK/AP-1/MMP-9 pathway by p21WAF1 expression in vascular smooth muscle cells. Cell Signal, 2014-12-08;27(3):470-8. 2014-12-08 [PMID: 25496832] (Western Blot, Human)
• Fliedl L, Manhart G, Kast F, Katinger H, Kunert R, Grillari J, Wieser M, Grillari-Voglauer R Novel human renal proximal tubular cell line for the production of complex proteins. J Biotechnol, 2014-02-16;176(0):29-39. 2014-02-16 [PMID: 24553072] (Western Blot, Human)
• DeNiro M, Al-Mohanna F, Alsmadi O, Al-Mohanna F The nexus between VEGF and NFkappaB orchestrates a hypoxia-independent neovasculogenesis. PLoS ONE, 2013-03-22;8(3):e59021. 2013-03-22 [PMID: 23533599] (Western Blot, Human)
• Alexandra Castilho, Laura Neumann, Pia Gattinger, Richard Strasser, Karola Vorauer-Uhl, Thomas Sterovsky et al. Generation of Biologically Active Multi-Sialylated Recombinant Human EPOFc in Plants PLoS ONE 2013-01-25 [PMID: 23372778] (Western Blot, Plant - Nicotiana benthamiana)
• Alexandra Castilho, Laura Neumann, Sasha Daskalova, Hugh S. Mason, Herta Steinkellner, Friedrich Altmann et al. Engineering of Sialylated Mucin-type O-Glycosylation in Plants* Journal of Biological Chemistry 2012-10-19 [PMID: 22948156]
• Peter Solár, Gabriela Hrčková, Lenka Varinská, Zuzana Solárová, Ján Kriška, Ivana Uhrínová et al. Location and the functionality of erythropoietin receptor(s) in A2780 cells Oncology Reports 2012-04-30 [PMID: 22552716] (Western Blot, Human)
• Gassmann M, Grenacher B, Rohde B, Vogel J Quantifying Western blots: pitfalls of densitometry. Electrophoresis, 2009-06-01;30(11):1845-55. 2009-06-01 [PMID: 19517440] (Western Blot, Bovine, Camelid, Canine, Equine, Feline, Human, Mouse, Porcine, Primate - Pongo (Orangutan), Proboscine (Elephant), Rat, Rhinoceros)
• Delanghe JR, Bollen M, Beullens M Testing for recombinant erythropoietin. Am. J. Hematol., 2008-03-01;83(3):237-41. 2008-03-01 [PMID: 17918255] (Western Blot, Human)

Bioinformatics

• Gene Symbol: EPO
• Uniprot:
  • P01588()