Asialofetuin

Manufactured by Merck Group

Sourced in United States

Asialofetuin is a glycoprotein derived from fetal calf serum. It is commonly used as a reference standard and positive control in various laboratory techniques and assays.

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Lab products found in correlation

37 protocols using asialofetuin

Quantifying Cell Proliferation via Live-Cell Imaging

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HEPG2 cells (100,000 cells/well in a 24-well plate) were treated with various concentrations of PIP or PIP-GalNAc. After treatments were added, cells were imaged every 4 hours for the indicated times in the IncuCyte S3 Live-Cell Analysis system using the Phase imaging channel and 10X objective setting. For co-incubation experiment with asialofetuin, 10 mg/ml of asialofetuin (Sigma Aldrich) was treated with either PIP or PIP-GalNAc.
The following analysis was used to quantify Phase Confluence (%) in the Incucyte software: Segmentation Adjustment = 1, Cleanup- Hole Fill (μm²) = 300, Filters- Area Minimum (μm²) = 150. No other constraints were selected.
Percent proliferation was then calculated by normalizing Phase Confluence (%) values. Specifically, at any given timepoint n in the treatment time course (t = 0 → t = n), the change in Confluence from t = 0 to t = n (ΔConfluence _{t = n}) was calculated for each well as follows: ΔConfluence _{t = n} of well x = (Confluence _{t = n} of well x) − (Confluence _{t = 0} of well x), where x represents any given well in the experiment. This ensures the value of each well is set to 0 at t = 0. The average ΔConfluence at the final time point of the untreated wells was set as the “Max Value” (equivalent to 100% Proliferation). Finally, data were normalized as follows:

% {Proliferation}_{t = n} of well x = (Δ {Confluence}_{t = n} of well x) / (M a x Value) .

Ahn G., Banik S.M., Miller C.L., Riley N.M., Cochran J.R, & Bertozzi C.R. (2021). LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation. Nature chemical biology, 17(9), 937-946.

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Quantifying RTB:IDUA Fusion Protein Activity

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An assay was developed to quantify RTB:IDUA fusion proteins that takes into account both RTB and IDUA activities. It is based on lectin-mediated affinity to glycoproteins by RTB and direct IDUA activity using 4MUI. Immulon 4HBX plates were coated with asialofetuin (Sigma-Aldrich) containing solution (300 μg/mL) in bicarbonate buffer (pH 9.5) followed by incubation at room temperature for 1 h. Following a washing step with 0.5% Tween-20 in PBS, 100 μL of diluted samples (1:20) in PBS + 1% BSA were added to the asialofetuin-coated plate and incubated for 1 h at room temperature. Following a second wash step, an aliquot of extracts from plants infiltrated with an empty vector (pBIB-Kan; 5 μl) was added to all test sample wells to control for components that may be present in plant crude extracts. A control crude sample containing an aliquot of the recombinant fusion crude extract (5 μl) was applied to unloaded wells in order to determine the activity emitted from any enzymatically active IDUA (with or without lectin binding activity). Reaction was initiated with 10 μl of substrate solution (0.1 M sodium acetate pH 4.8, 1 mM sodium metabisulphite, 3.5 mg/ml BSA and 0.75 mM 4MUI) and incubated for 60 min at 37 °C. Fluorometric determination for detecting and quantifying fluorescent breakdown of the substrate was performed as described above for IDUA enzymatic assays.

Acosta W., Ayala J., Dolan M.C, & Cramer C.L. (2015). RTB Lectin: a novel receptor-independent delivery system for lysosomal enzyme replacement therapies. Scientific Reports, 5, 14144.

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Quantifying Cell Proliferation via Live-Cell Imaging

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% {Proliferation}_{t = n} of well x = (Δ {Confluence}_{t = n} of well x) / (M a x Value) .

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Carboxybetaine Thiol Synthesis and Characterization

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Potassium hexacyanoferrate(III)
(ferricyanide), Potassium hexacyanoferrate(II) trihydrate (ferrocyanide),
potassium chloride, phosphate buffer saline tablets, sulfuric acid,
sodium hydroxide, N-hydroxysuccinimide (NHS), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide
hydrochloride (EDC), Ricinus communis agglutinin (RCA, recognizing galactose, caution: handle
with special care because it is a biological toxin), fetuin
(FET, contains 8.7% of sialic acid), and asialofetuin (ASF, contains
≤0.5% of sialic acid) were purchased from Sigma-Aldrich (U.S.). Sambucus nigra agglutinin type I (SNA, recognizing
sialic acid) lectin from Sambucus nigra was purchased from EYLabs (U.S.). Ethanol for UV/vis spectroscopy
(ultrapure) was purchased from Slavus (Slovakia). Biotinylated lectins
and a carbo-free blocking solution were purchased from Vector Laboratories
(U.S.). CF555-streptavidin fluorescent label was purchased from Biotium
(U.S.). All solutions were filtered prior to use (using 0.2 μm
sterile filters) and prepared in ultrapure distilled water (DW). The
synthesis of the carboxybetaine thiol (CB) together with its spectral
analysis is provided in the Supporting Information, and the key steps in the synthesis are shown in Scheme 1.

Bertok T., Šedivá A., Filip J., Ilcikova M., Kasak P., Velic D., Jane E., Mravcová M., Rovenský J., Kunzo P., Lobotka P., Šmatko V., Vikartovská A, & Tkac J. (2015). Carboxybetaine Modified Interface for Electrochemical Glycoprofiling of Antibodies Isolated from Human Serum. Langmuir, 31(25), 7148-7157.

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Glycoprotein Analysis: Enzymatic Approaches

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Dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate (ABC), ammonium acetate (AA), ammonium hydroxide, ribonuclease B from bovine pancreas (RNase B), fetuin from fetal calf serum (bovine fetuin), asialofetuin from fetal calf serum (asialofetuin) and alpha-1 acid glycoprotein from human plasma (AGP) were purchased from Sigma-Aldrich (St. Louis, MO). Murine IgG1 (Intact mAb Mass Check Standard) was obtained from Waters Corporation (Milford, MA). α2–3 neuraminidase, α2–3,6,8,9 neuraminidase, β1–3 galactosidase, β1–4 galactosidase and β1–3,4 galactosidase were acquired from New England Biolabs (Ipswich, MA). Mass spectrometry grade Trypsin/Lys-C mixture, sequencing grade chymotrypsin and sequencing grade endoprotease Glu-C (Glu-C) were obtained from Promega (Madison, WI). HPLC grade water was acquired from Mallinckrodt Chemicals (Phillipsburg, NJ). HPLC grade acetonitrile was purchased from Fisher Scientific (Fair Lawn, NJ).

Zhu R., Huang Y., Zhao J., Zhong J, & Mechref Y. (2020). Isomeric Separation of N-Glycopeptides Derived from Glycoproteins by Porous Graphitic Carbon (PGC) LC-MS/MS. Analytical chemistry, 92(14), 9556-9565.

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Platelet Depletion for Malaria Sporozoite Immunity

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Mice were immunized intravenously (i.v.) via tail vein injection with 5×10⁴P. berghei CS^5M (37 (link)) sporozoites dissected by hand from the salivary glands of Anopheles stephensi mosquitos generated in-house within a quarantine approved facility. Prior to immunization, sporozoites were irradiated at 200kRad of gamma radiation and delivered to each subject. Mice were monitored for the following 21 days for any sign of breakthrough infection both through behavioural changes and blood smear analysis. Platelet depletion of experimental mice was achieved using monoclonal antibodies specific for either glycoprotein Ibα (anti-GPIbα; R300 polyclonal- Emfret) or the αIIbβ3 integrin (anti-αIIbβ3; Leo.H4-Emfret) at a concentration of 20μg per mouse in PBS delivered i.v. via the tail vein. Platelet depletion occurred within 30 mins post-injection; however, mice were monitored for 60 mins for adverse reactions or excessive bleeding. Control mice received 20μg of an isotype control antibody diluted in PBS and delivered i.v via the tail vein. For lectin blockade, mice were treated with Asialo Fetuin or Fetuin (Sigma-Aldrich) i.v at concentrations ranging from 1mg/mouse to 3mg/mouse diluted in cold PBS and delivered prior to adoptive transfer studies.

O’Connor J.H., McNamara H.A., Cai Y., Coupland L.A., Gardiner E.E., Parish C.R., McMorran B.J., Ganusov V.V, & Cockburn I.A. (2022). Interactions with asialo-glycoprotein receptors and platelets are dispensable for CD8+ T cell localization in the murine liver. Journal of immunology (Baltimore, Md. : 1950), 208(12), 2738-2748.

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Serum FSH Kinetics of Follitropin Analogs

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Identical volumes (0.5 mL) and concentrations (4 µg/mL) of follitropin delta and follitropin alfa were injected IV into male Sprague–Dawley rats weighing 80–85 g (Harlan) in the presence and absence of asialofetuin (Sigma). Animals (2 per treatment group) were bled at specific time points (0.25, 1, 2 and 4 h post injection), and sera were tested for FSH concentration by ELISA in duplicates (DRG Instruments GmbH, Marburg, Germany). For the ELISA, 100 µL of anti-FSH enzyme conjugate was added to samples, and plates were incubated for 30 min at room temperature. Contents of wells were removed and rinsed 5 times with water, inverting plates to dry. Substrate solution was added and incubated 5 min at room temperature before adding 50 µL of stop solution, and absorbance was read at 450 nM. The anti-human FSH antibody used in the ELISA for the detection of recombinant human FSH proteins displayed no measureable interference from rat or mouse serum (data not shown). Results in the presence and absence of ASF were compared using a Student’s t-test at P < 0.05.

Koechling W., Plaksin D., Croston G.E., Jeppesen J.V., Macklon K.T, & Andersen C.Y. (2017). Comparative pharmacology of a new recombinant FSH expressed by a human cell line. Endocrine Connections, 6(5), 297-305.

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Quantifying Dual Bioactivity in Fusion Proteins

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The “Dual” assay is a combination of a lectin binding [21 (link)] and β-gal activity assays and was developed to directly quantify products having both active RTB and active β-gal in a fusion product. The fusion protein is captured based on the affinity of RTB to bind glycans on the surface of the asialofetuin glycoprotein. First, asialofetuin (Sigma) is bound to the surface of an Immulon 4HBX 96 well plate (Thermo-Fisher) at a concentration of 3 μg/ml. After RTB is bound to asialofetuin, the enzymatic activity of the β-gal is measured as described in the β-gal assay (2.4). The dual bioactivity assay was modified from a previous study [24 (link)]. Briefly, 100 μl of crude extract, 1:100 dilution in PBS containing 1% BSA, was loaded onto Immulon 96-well plate. After incubating for 1 h at room temperature, unbound proteins were washed away and 20 μl of substrate solution (1 mM 4MUGal in 100 mM sodium acetate buffer, pH 4.3) was added and incubated at 37°C for 30 min in the dark. The reaction was stopped by adding 280 μl of stop solution (see 2.4). The same procedure was done with crude extract from plants infiltrated with an empty vector to serve as a blank. The standard curve and fluorescence reading were all done as described in section 2.4. For both activity and Dual assay, the activity of the proteins is defined as nmol/min or unit of activity (U), which is equal to nmol/min.

Condori J., Acosta W., Ayala J., Katta V., Flory A., Martin R., Radin J., Cramer C.L, & Radin D.N. (2015). Enzyme replacement for GM1-gangliosidosis: Uptake, lysosomal activation, and cellular disease correction using a novel β-galactosidase:RTB lectin fusion. Molecular genetics and metabolism, 117(2), 199-209.

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Influenza Virus HA Protein Purification

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rHAs of influenza A (H1N1 and H5N1) and B viruses were purchased from Protein Science Inc (Meriden, CT) and rHAs of influenza A H7N9 viruses were purchased from Sino Biological (Beijing, China). Bovine and human fetuin, asialofetuin and all other chemicals were purchased from Sigma (St Louis, MO).

Jiang L, & Eichelberger M.C. (2015). Evaluation of Epic® label-free technology to quantify functional recombinant hemagglutinin. Biological Procedures Online, 17, 7.

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Glycan Characterization Protocol

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Cytidine 5′-diphosphate (CDP), cytidine, adenosine 5′-diphosphate (ADP), and 6`-sialyllactose were purchased from GeneChem (Daejeon, Korea). Peptide-N-glycosidase F (PNGase F) and graphitized carbon column were obtained from New England Biolabs (Ipswich, MA, USA) and Alltech (Lexington, MA, USA) respectively. Asialo, galactosylated, biantennary oligosaccharide (G2 glycan), calf intestinal alkaline phosphatase, and biotinylated Sambucus nigra (SNA) lectin were purchased from Prozyme (Hayward, CA), Takara (Tokyo, Japan), and Ey Laboratories (San Mateo, CA, USA) respectively. Solvents for high-performance liquid chromatography (HPLC) including acetonitrile and water were purchased from Burdick and Jackson (Muskegon, MI, USA). 2-Aminobenzoic acid (AA), sodium cyanoborohydride, acetic acid, tetrahydrofuran, triethylamine, trifluoroacetic acid (TFA), CMP-NeuAc, adenosine 5′-triphosphate (ATP), cytidine 5′-triphosphate (CTP), cytidine 5′-monophosphate (CMP), GDP-galactose, bovine β(1,4)-galactosyltransferase, asialofetuin and other reagents (unless stated otherwise) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Kang J.Y., Lim S.J., Kwon O., Lee S.G., Kim H.H, & Oh D.B. (2015). Enhanced Bacterial α(2,6)-Sialyltransferase Reaction through an Inhibition of Its Inherent Sialidase Activity by Dephosphorylation of Cytidine-5'-Monophosphate. PLoS ONE, 10(7), e0133739.

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