Ultralink hydrazide resin

Manufactured by Thermo Fisher Scientific

Sourced in United States

UltraLink hydrazide resin is a matrix-based affinity chromatography resin designed for the immobilization and purification of oxidized glycoproteins. The resin features a hydrazide functional group that covalently binds to the carbonyl groups of glycans, enabling the capture and enrichment of glycoproteins from complex samples.

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Lab products found in correlation

7 protocols using ultralink hydrazide resin

Haptoglobin Purification and Analysis

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Haptoglobin was purified from a 20-μL aliquot of serum for each patient by using an HPLC-based antibody-immobilized column developed in-house as previously reported [38 (link)]. Briefly, the mouse anti-human haptoglobin antibody was covalently immobilized on the UltraLink hydrazide resin (Thermo Scientific, Rockford, IL) and then packed into a PEEK column (4.6 mm × 50 mm). The immunoaffinity purification of haptoglobin was performed on a Beckman Coulter ProteomeLab PPS system (Fullerton, CA) based on the HPLC platform developed previously [38 (link)]. The bound haptoglobin fraction was eluted with stripping buffer (0.1 M Glycine, pH 2.5) and then immediately neutralized with 0.1 M Tris-HCl (pH 8.0). Subsequently, the enriched haptoglobin was desalted using a YM-3 centrifugal filter device (Millipore, Billerica, MA) by buffer exchange with water for three times and then dried down in a SpeedVac concentrator (Thermo). Before glycan release, the purity of the eluted haptoglobin was confirmed by 1D SDS-PAGE followed by silver staining using ProteoSilver™ Plus Silver Stain Kit (Sigma).

Huang Y., Zhou S., Zhu J., Lubman D.M, & Mechref Y. (2017). LC-MS/MS Isomeric Profiling of Permethylated N-Glycans Derived from Serum Haptoglobin of Hepatocellular Carcinoma (HCC) and Cirrhotic Patients. Electrophoresis, 38(17), 2160-2167.

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Glycan Isolation and Purification

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Glycan(s) of interest

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Sodium bicarbonate buffer: 38 g L⁻¹ NaHCO₃, pH 8.5

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Acetyl acetone

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Ethyl acetate

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Dowex 50WX8 strong acid ion exchange resin (Sigma–Aldrich, cat. 217492)

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Ultralink hydrazide resin (Thermo Scientific, cat. 53149)

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Pyridine

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Sodium azide

Note: Materials lists include only non-standard items. Common laboratory ware and equipment such as water baths are assumed to be available.

Van Tassell M.L., Price N.P, & Miller M.J. (2014). Glycan-specific whole cell affinity chromatography: A versatile microbial adhesion platform. MethodsX, 1, 244-250.

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Haptoglobin Glycan Analysis via PNGase F

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N-Glycosidase F (PNGase F) was purchased from New England Biolabs (Ipswich, MA). Neuraminidase, sodium hydroxide, methyl iodide, β-mercaptoethanol, chloroform, dimethyl sulfoxide (DMSO), HPLC-grade acetonitrile (ACN), and water and the 96-well vacuum manifold were purchased from Sigma-Aldrich (St. Louis, MO). The MALDI matrix, 2,5-dihydroxybenzoic acid (2,5-DHB), the UltraLink hydrazide resin, the Zeba Spin Desalting column, and the SPE 96-well plate packed with 100% porous graphitic carbon (PGC) were purchased from Thermo Scientific (Rockford, IL). The 96-well SpinColumn plate was purchased from Harvard Apparatus (Holliston, MA). Antihuman haptoglobin antibody (CatLog No. ab13429) and a human haptoglobin standard were purchased from Abcam (Cambridge, MA). The empty PEEK column was purchased from MicroSolv Technology Corp. (Eatontown, NJ).

Zhu J., Wu J., Yin H., Marrero J, & Lubman D.M. (2015). Mass Spectrometric N-Glycan Analysis of Haptoglobin from Patient Serum Samples Using a 96-Well Plate Format. Journal of proteome research, 14(11), 4932-4939.

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Haptoglobin Purification from Patient Serum

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Haptoglobin (Hp) was purified from 20 μL of patient serum using an antibody-immobilized HPLC column which has a recovery of 40–50% of Hp from serum samples as described previously.^{37 (link)} The immunopurification was performed on a Beckman Coulter HPLC system (Fullerton, CA) with a PEEK column (4.6 mm × 50 mm) packed in-house with the UltraLink hydrazide resin (Thermo Scientific, Rockford, IL) conjugated with an antihuman Hp antibody (Abcam, Cambridge, MA). The eluted haptoglobin fraction was desalted using a 4 mL YM-3 centrifugal device by buffer exchange 3 times with deionized water and then dried down in a SpeedVac concentrator (Thermo). The HPLC peak area of the eluted Hp fraction was measured and compared among patients (Supplemental Table S1), where SDS-PAGE gel analysis of the Hp eluent showed the purity of haptoglobin fraction (Supplemental Figure S1). One-tenth of the Hp eluent was loaded onto the gel, with 0.3 μg of a haptoglobin standard protein (Abcam) as a reference. On the basis of the gel result, the amount of haptoglobin used for the subsequent trypsin digestion was estimated to be around 3 μg.

Zhu J., Chen Z., Zhang J., An M., Wu J., Yu Q., Skilton S.J., Bern M., Sen K.I., Li L, & Lubman D.M. (2018). Differential Quantitative Determination of Site-Specific Intact N-Glycopeptides in Serum Haptoglobin between Hepatocellular Carcinoma and Cirrhosis Using LC-EThcD-MS/MS. Journal of proteome research, 18(1), 359-371.

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Purification of B. pseudomallei OPS Antibodies

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To facilitate purification of specific antibodies against B. pseudomallei OPS, type A LPS was extracted from the select agent excluded strain B. pseudomallei RR2808 using a modified hot phenol method (37 (link), 38 (link)). Purified OPS antigens were then obtained by acid hydrolysis and size exclusion chromatography essentially as previously described (37 (link), 39 (link)). The OPS was then oxidized with sodium metaperiodate as previously described (37 (link)) and conjugated to UltraLink Hydrazide resin (Thermo Scientific, USA) via reductive amination following the manufacturer’s instructions. The resulting OPS-coupled resin was stored at 4°C until use.

Pumpuang A., Paksanont S., Burtnick M.N., Brett P.J, & Chantratita N. (2022). Functional Activities of O-Polysaccharide and Hemolysin Coregulated Protein 1 Specific Antibodies Isolated from Melioidosis Patients. Infection and Immunity, 90(11), e00214-22.

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Purification of PEG-Qdot655 Nanoparticles

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PEG-Qdot655 nanoparticles were purified by affinity chromatography on the anti-PEG antibody resin columns. Briefly, 5 mg of 3.3 or 2B5 anti-PEG antibodies in coupling buffer (0.1 M sodium acetate, 0.15 M sodium chloride, pH 5.5) was reacted with 10 mM sodium meta-periodate (Thermo Scientific Pierce) in the dark at room temperature for 30 min. sodium meta-periodate was removed on a Zeba™ Spin desalting column and the oxidized antibodies were then incubated with 1 mL of UltraLink® Hydrazide Resin containing 0.1 M aniline (Thermo Scientific) at room temperature for 4 h. The anti-PEG resin was packed into a column and washed with PBS 3 times. Five hundred microliters of PEG-Qdot655 solution (32 nM in PBS) was loaded into the 3.3 or 2B5 anti-PEG columns at 4°C. The columns were washed with cold PBS and bound PEG-Qdot655 nanoparticles were eluted with 37°C PBS or 100 mM citrate buffer (pH = 3). The particles were transferred into a Nunc F96 MicroWell black polystyrene plate (200 μL/well) and the fluorescence of PEG-Qdot655 (excitation/emission: 450 nm/660 nm) was detected on an IVIS 200 optical imaging system (Xenogen).

Su Y.C., Al-Qaisi T.S., Tung H.Y., Cheng T.L., Chuang K.H., Chen B.M, & Roffler S.R. (2014). Mimicking the germinal center reaction in hybridoma cells to isolate temperature-selective anti-PEG antibodies. mAbs, 6(4), 1069-1083.

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Peptide Synthesis and Fluorescent Labeling

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Peptide 1D4 (TETSQVAPA) was synthesized at the Keck Biotechnology Resource Laboratory at Yale University. Two fluorescently labeled peptides, GLP-1-(7–37) and Ex-4, were also obtained from there with the E21K and L21K mutations, respectively for lysine conjugation with 5(6)-carboxyfluorescein (FAM). The following materials were purchased from indicated sources: peptide GLP-1-(7–37) from GL Biochem (Shanghai) Ltd; Ex-4 from Abcam; BODIPY-FL-GTPγS from Invitrogen; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) from Avanti Polar Lipids; n-dodecyl-β-D-maltopyranoside (DDM) from Anatrace; Bio-Beads SM-2 from Bio-Rad; and Rho 1D4 purified monoclonal antibody from University of British Columbia. The antibody was coupled to UltraLink Hydrazide resin purchased from Thermo Scientific [36 (link), 37 (link)]. All other chemicals were analytical grade obtained from Sigma or American Biochemicals. Membrane scaffold protein (MSP1E3D1) was expressed and purified as described previously [33 , 35 (link)].

Cai Y., Liu Y., Culhane K.J., DeVree B.T., Yang Y., Sunahara R.K, & Yan E.C. (2017). Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor. PLoS ONE, 12(6), e0179568.

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