Recombinant Human RBP4 Protein, CF

• Reactivity: Hu
• Applications: Binding Activity
• Format: Carrier-Free

Recombinant Human RBP4 Protein, CF Summary

• Details of Functionality: Measured by its ability to bind all-trans retinoic acid. The binding of retinoic acid results in the quenching of Trp fluorescence in RBP4. >1.0 µM all-trans retinoic acid is bound under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human RBP4/Retinol-Binding Protein 4 protein
  Glu19-Leu201, with a C-terminal Ile and 6-His tag
• Accession #: P02753
• N-terminal Sequence: Glu19
• Protein/Peptide Type: Recombinant Enzymes
• Gene: RBP4
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Binding Activity
• Theoretical MW: 22 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 22 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Reconstitution Instructions: Reconstitute at 500 μg/mL in sterile 50 mM Tris, 10 mM CaCl₂ and 150 mM NaCl, pH 7.5.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5 (TCN)
  • Recombinant Human RBP4/Retinol‑Binding Protein 4 (rhRBP4) (Catalog # 3378-LC)
  • 95% Ethanol
  • Retinoic Acid (Tocris, Catalog # 0695), 50 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhRBP4 to 49 µg/mL in Assay Buffer.
  2. Make serial dilutions of retinoic acid in 95% ethanol at 100, 30, 10, 3 and 1 µM.
  3. The formation of Protein/Retinol binding in microtubes:
  1. Mix 112.5 µL of 49 µg/mL rhRBP4 and 12.5 µL of retinoic acid serial dilutions in microtubes.
  2. For a blank, mix 112.5 µL of 49 µg/mL rhRBP4 and 12.5 µL of 95% ethanol in a microtube.
  4. Incubate reaction tubes at room temperature for 30 minutes.
  5. In a plate load 100 µL of the reaction mixtures and blank.
  6. Read at excitation and emission wavelengths of 280 nm and 340 nm (top read), respectively, in endpoint mode.
  7. Calculate concentration at which 50% quenching of rhRBP4 is achieved by plotting raw RFUs vs. concentration of retinoic acid with 4‑PL fitting. Use this value to estimate the concentration of retinoic acid fully bound by the rhRBP4.
  Per Well:
  • rhRBP4: 4.41 µg
  • Retinoic Acid Curve: 10, 3, 1, 0.3 and 0.1 µM

Notes

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

Alternate Names for Recombinant Human RBP4 Protein, CF

• interstitial
• Plasma retinol-binding protein
• RBP4
• retinol binding protein 4, plasma
• RetinolBinding Protein 4
• retinol-binding protein 4, plasma

Background

Retinol (also known as vitamin A) is unstable and insoluble in the aqueous solution (1, 2). However, retinol becomes quite stable and soluble in plasma due to its tight interaction with retinol-binding protein 4 (RBP4), also known as plasma retinol-binding protein. A prototypic member of the lipocalin superfamily, RBP4 has a beta -barrel structure with a well-defined cavity. It is secreted from the liver, a process requiring the availability of retinol. RBP4 delivers retinol from the liver to the peripheral tissues. In plasma, the RBP4-retinol complex interacts with transthyretin (TTR), also known as thyroxine-binding protein and prealbumin. The retinol-RBP4-TTR complex prevents the loss of RBP4 by filtration through the kidney and increases the stability of the retinol-RBP4 complex. Defects in RBP4 cause retinol-binding protein deficiency, which affects night vision.

1. Zanotti, G. and R. Berni. (2004) Vitamins and Hormones 69:271.
2. Newcomer, M.E. and D.E. Ong. (2000) Biochim. Biophys. Acta 1482:57.

Publications for RBP4/Retinol-Binding Protein 4 (3378-LC)(2)

Publications using 3378-LC

• T Muraguchi, D Nanba, EK Nishimura, T Tashiro IGF-1R deficiency in human keratinocytes disrupts epidermal homeostasis and stem cell maintenance J. Dermatol. Sci., 2019-05-11;0(0):. 2019-05-11 [PMID: 31122679] (Bioassay, Human)
• Schipper HS, De Jager W, van Dijk ME, Meerding J, Zelissen PM, Adan RA, Prakken BJ, Kalkhoven E A multiplex immunoassay for human adipokine profiling. Clin. Chem., 2010-06-08;56(0):1320. 2010-06-08 [PMID: 20530731] (ELISA (Standard))

Bioinformatics

• Gene Symbol: RBP4
• Uniprot:
  • P02753()