Midkine Antibody [Unconjugated]

Western blot shows lysates of SH-SY5Y human neuroblastoma cell line. PVDF membrane was probed with 2 µg/mL of Goat Anti-Human Midkine Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-258-PB) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF017). A specific band was detected for Midkine at approximately 17 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Midkine was detected in immersion fixed paraffin-embedded sections of human prostate using Goat Anti-Human Midkine Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-258-PB) at 10 µg/mL overnight at 4 °C. Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (CTS013). Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown CTS008) and counterstained with hematoxylin (blue). Specific staining was localized to stromal cell cytoplasm. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Midkine was detected in immersion fixed paraffin-embedded sections of human ovarian array using Goat Anti-Human Midkine Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-258-PB) at 10 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; CTS008) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Simple Western lane view shows lysates of SH‑SY5Y human neuroblastoma cell line, loaded at 0.2 mg/mL. A specific band was detected for Midkine at approximately 26 kDa (as indicated) using 100 µg/mL of Goat Anti-Human Midkine Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-258-PB) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Western blot shows lysates of HAP1 human near-haploid cell line untreated (-) or treated (+) with 3.0 ug/ml Brefeldin A for 18 hr and Midkine knockout HAP1 cell line (KO). Nitrocellulose membrane was probed with 2 µg/mL of Goat Anti-Human Midkine Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-258-PB) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody. A specific band was detected for Midkine at approximately 11 kDa (as indicated) in the parental HAP1 cell line, but is not detectable in knockout HAP1 cell line. The Ponceau stained transfer of the blot is shown. This experiment was conducted under reducing conditions. Image, protocol, and testing courtesy of YCharOS Inc. See ycharos.com for additional details.

Immunoprecipitation was performed on concentrated culture media of HAP1 human near-haploid cell line using 2.0 μg of Mouse Anti-Human Midkine Monoclonal Antibody (Catalog # AF-258-PB) pre-coupled to protein G or protein A beads. Immunoprecipitated Midkine was detected with Rabbit Anti-Human Midkine Monoclonal Antibody (NBP2-66948 ) at 1/500. The Ponceau stained transfers of each blot are shown. SM=10% starting material; UB=10% unbound fraction; IP=immunoprecipitated. Image, protocol, and testing courtesy of YCharOS Inc. (ycharos.com).

• Reactivity: Hu
• Applications: WB, Simple Western, IHC, IP, KO
• Clonality: Polyclonal
• Host: Goat
• Conjugate: Unconjugated
• Concentration: LYOPH

Midkine Antibody [Unconjugated] Summary

• Immunogen: E. coli-derived recombinant human Midkine
  Lys23-Asp143
  Accession # P21741
• Specificity: Detects human Midkine in direct ELISAs and Western blots. In direct ELISAs, approximately 10% cross-reactivity with recombinant mouse Midkine is observed, and less than 1% cross-reactivity with recombinant human Pleiotrophin is observed.
• Source: N/A
• Isotype: IgG
• Clonality: Polyclonal
• Host: Goat
• Gene: MDK
• Purity Statement: Antigen Affinity-purified

Applications/Dilutions

• Dilutions:
  • Immunohistochemistry 5-15 ug/mL
  • Knockout Validated
  • Simple Western 100 ug/mL
  • Western Blot 2 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 reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.2 mg/mL in sterile PBS.

Notes

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

Alternate Names for Midkine Antibody [Unconjugated]

• Amphiregulin-associated protein
• ARAP
• MDK
• MEK
• Midgestation and kidney protein
• midkine (neurite growth-promoting factor 2)
• Midkine
• MK1
• MKARAP
• NEGF2
• NEGF2FLJ27379
• Neurite outgrowth-promoting factor 2
• Neurite outgrowth-promoting protein

Background

Midkine (MK) is a 15 kDa heparin-binding molecule originally cloned during a search for genes preferentially transcribed during retinoic acid (RA)-induced differentiation. Midkine belongs to a family of neurotrophic and developmentally-regulated heparin-binding molecules consisting of midkine, pleiotrophin (PTN/HBNF/OSF-1/HNGF-8) and the avian midkine homolog, RI-HB (for retinoic acid-inducible heparin-binding protein).

Midkine is a highly basic, nonglycosylated polypeptide that contains five intrachain disulfide bonds. The predicted molecular weight is approximately 13.3 kDa, based on a mature peptide length of 118 amino acid residues in the mouse and 121 amino acid residues in the human. Across species, MK shows 87% identity between the human and murine proteins. Between family members, human MK is approximately 50% identical to human PTN, with conservation of all 10 cysteines. Initial structure-function studies indicate that the C-terminal half of MK contains the principal heparin-binding site plus the molecule’s antigenicity and neurite-promoting sequences; while both the C- and N-termini are necessary for the molecule’s neurotrophic effects. Cells known to produce MK include endothelial cells, fetal astrocytes, renal proximal tubule epithelial cells and Wilms’ (kidney) tumor cells. MK has also been identified in the senile plaques of patients with Alzheimer’s disease. The pattern of expression of midkine during development strongly suggests a role for this factor both in epithelial-mesenchymal interactions and in development of the nervous system.

1. Bohlen, P. and I. Kovesdi (1991) Prog. Growth Factor Res. 3:143.
2. Muramatsu, T. (1993) Int. J. Dev. Biol. 37:183.

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 Midkine Antibody (AF-258-PB)(9)

Publications using AF-258-PB

• Ayoubi, R;Southern, K;Laflamme, C;NeuroSGC/YCharOS Collaborative Group, ; A guide to selecting high-performing antibodies for human Midkine for use in Western blot and immunoprecipitation F1000Research 2024-08-01 [PMID: 39092005] (Immunoprecipitation, Western Blot, Human)
• Yun Zhu, Alfred K.Y. Lam, Daisy K.Y. Shum, Di Cui, Jun Zhang, Dong Dong Yan et al. Significance of serglycin and its binding partners in autocrine promotion of metastasis in esophageal cancer Theranostics 1/1/2021 [PMID: 33456569]
• DH Shin, JY Jo, SH Kim, M Choi, C Han, BK Choi, SS Kim Midkine Is a Potential Therapeutic Target of Tumorigenesis, Angiogenesis, and Metastasis in Non-Small Cell Lung Cancer Cancers (Basel), 2020-08-24;12(9):. 2020-08-24 [PMID: 32847073] (IHC, Human, Mouse)
• Bing Bai, Xusheng Wang, Yuxin Li, Ping-Chung Chen, Kaiwen Yu, Kaushik Kumar Dey et al. Deep Multilayer Brain Proteomics Identifies Molecular Networks in Alzheimer’s Disease Progression Neuron 3/1/2020 [PMID: 31926610]
• Imad Tarhoni, Cristina L. Fhied, Mark Pool, Michael J. Liptay, Philip Bonomi, Christopher W. Seder et al. Development of bead based multiplexed immunoassay for evaluation of midkine, syndecan-1, and ANGPTL4 in patient serum Journal of Immunoassay and Immunochemistry 2/2/2018 [PMID: 29309212]
• Marchionini DM, Lehrmann E, Chu Y, He B, Sortwell CE, Becker KG, Freed WJ, Kordower JH, Collier TJ Role of heparin binding growth factors in nigrostriatal dopamine system development and Parkinson's disease. Brain Res., 2007-02-22;1147(0):77-88. 2007-02-22 [PMID: 17368428] (Western Blot, Rat)
• Li F, Shetty AK, Sugahara K Neuritogenic activity of chondroitin/dermatan sulfate hybrid chains of embryonic pig brain and their mimicry from shark liver. Involvement of the pleiotrophin and hepatocyte growth factor signaling pathways. J. Biol. Chem., 2006-12-04;282(5):2956-66. 2006-12-04 [PMID: 17145750] (Neutralization, Mouse)
• Kang HC, Kim IJ, Park HW, Jang SG, Ahn SA, Yoon SN, Chang HJ, Yoo BC, Park JG Regulation of MDK expression in human cancer cells modulates sensitivities to various anticancer drugs: MDK overexpression confers to a multi-drug resistance. Cancer Lett., 2006-04-27;247(1):40-7. 2006-04-27 [PMID: 16644108] (Western Blot, Human)
• Said EA, Krust B, Nisole S, Svab J, Briand JP, Hovanessian AG The anti-HIV cytokine midkine binds the cell surface-expressed nucleolin as a low affinity receptor. J. Biol. Chem., 2002-07-29;277(40):37492-502. 2002-07-29 [PMID: 12147681] (ICC, Western Blot, Human)

Bioinformatics

• Gene Symbol: MDK
• Uniprot:
  • P21741()