Sulfolink

Manufactured by Thermo Fisher Scientific

SulfoLink is a crosslinking reagent used for the covalent immobilization of proteins and other molecules onto agarose beads. It provides a stable thioether linkage between the target molecule and the agarose support.

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Lab products found in correlation

Bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Bis tris gel, by Thermo Fisher Scientific (1 mentions) Aminolink, by Thermo Fisher Scientific (1 mentions) Bca protein assay, by Thermo Fisher Scientific (1 mentions)

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SulfoLink Coupling Resin (45 citations) SulfoLink resin (21 citations) SulfoLink Immobilization Kit for Peptides (16 citations) SulfoLink kit (11 citations) SulfoLink Immobilization Kit (7 citations) SulfoLink Immobilization Kit for Proteins (5 citations) SulfoLink Coupling Gel (4 citations) Sulfolink columns (4 citations) Sulfolink resin column (2 citations) Sulfolink agarose (2 citations)

7 protocols using sulfolink

Purification and Analysis of Anti-α-Syn Antibodies

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Anti-hα-Syn antibodies from sera of mice immunized with
PV-1947D, PV-1948D and PV-1949D epitope vaccines were purified by an affinity
column (SulfoLink, ThermoFisher Sci.) using an immobilized
α-Syn_85–99-C,
α-Syn_109–126-C and
α-Syn_126–140-C peptides (GenScript),
respectively, as we previously described(Mamikonyan et al., 2007 (link)). Purified antibodies were analyzed via
10% Bis-Tris gel (ThermoFisher Sci.), and the concentrations were
determined using a BCA protein assay kit (ThermoFisher Sci.).

Davtyan H., Zagorski K., Petrushina I., Kazarian K., Goldberg N.R., Petrosyan J., Blurton-Jones M., Masliah E., Cribbs D.H., Agadjanyan M.G, & Ghochikyan A. (2017). MultiTEP Platform-based DNA Vaccines for alpha-Synucleinopathies: Pre-clinical Evaluation of Immunogenicity and Therapeutic Potency. Neurobiology of aging, 59, 156-170.

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Immunogenic Epitope Identification and Antibody Generation

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The amino acid sequence of rMBPPfCox11Ct was used to identify immunogenic epitopes with Predict7 software [40 (link)]. The peptide sequence KIQF(Abu)F(Abu)EEQMLNAKEEM where internal cysteines were replaced with alpha aminobutyric acid (Abu), was synthesized by GeneScript, USA. The C-terminal cysteine residue was added for m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) coupling to rabbit serum albumin [41 (link)]. Two Hy-line brown laying hens were immunized with the peptide-carrier conjugate equivalent to 200 µg peptide or 50 µg affinity purified rMBPPfCox11Ct per immunization, emulsified with Freund’s complete adjuvant for the first immunization and Freund’s incomplete adjuvant for three subsequent immunizations at 2-week intervals. Chicken IgY from the yolks of eggs collected 4–16 weeks after the first immunization, was isolated using the PEG6000 precipitation method [42 (link)]. The anti-peptide IgY and anti-rMBPPfCox11Ct IgY was affinity purified using a peptide or rMBPPfCox11Ct affinity column prepared by coupling the respective molecules to a Sulfolink^™ or Aminolink^™ resin according to the manufacturer’s instructions (Thermo Fisher Scientific).

Salman A.A, & Goldring J.P. (2022). Expression and copper binding characteristics of Plasmodium falciparum cytochrome c oxidase assembly factor 11, Cox11. Malaria Journal, 21, 173.

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Generation and Validation of Anti-VXN Antibody

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A rabbit polyclonal antibody was generated directed against a peptide (EYIGASNCAFEDD) in the conserved C-terminus of the protein. The antibody was affinity purified using a SulfoLink™ column (ThermoFisher Scientific cat# 20401) according to manufacturer’s protocols. To confirm that the affinity purified antibody recognizes both Xenopus and mouse VXN protein, western blot analysis of in vitro translated Xenopus vxn-3′myc and mouse 5′myc-vxn was performed using 1:400 of anti-Vxn antibody and confirmed by using anti-MYC 9E10 antibody to monitor Vxn 3′myc levels (Fig. S1B). Protein levels were quantified based on BCA protein assay (ThermoFisher Scientific) and 30 μg of total lysates were loaded per lane.

Moore K.B., Logan M.A., Diri I.A., Roberts J.M., Steele M, & Vetter M.L. (2018). C8orf46 homolog encodes a novel protein Vexin that is required for neurogenesis in Xenopus laevis. Developmental biology, 437(1), 27-40.

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Polyclonal Antibody Generation and Purification

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A more antigenic peptide corresponding to residues 22–46 of predicted protein (ARSSSYSGEYGSGGGKRFSHSGNQL) was synthesized at the scale of 5 mg. The peptide was used to immunize a rabbit and generate over 100 ml of antiserum by a standard immunization protocol (Rockland Inc., Limerick, PA). The polyclonal antibody from antiserum was purified through a peptide-bound affinity column (SulfoLink, Thermo Fisher Scientific Inc., Rockford, IL). This purified polyclonal antibody was used for western blot and immunohistochemistry (IHC) analysis.

Meng H., Li W., Boardman L.A, & Wang L. (2018). Loss of ZG16 is associated with molecular and clinicopathological phenotypes of colorectal cancer. BMC Cancer, 18, 433.

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Rabbit Polyclonal Antibody to Pig Chitinase

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Rabbit polyclonal antibodies to pig Chia was generated by Eurofins Genomics. Cys-peptides were conjugated through the added N-terminal cysteine to keyhole limpet hemocyanin (KLH). Sera from immunized rabbits were affinity-purified using the antigen with Cys (pig Chia, CMREAFEQEAKQTK) coupled to Sulfolink (Thermo Fisher Scientific).

Tabata E., Kashimura A., Uehara M., Wakita S., Sakaguchi M., Sugahara Y., Yurimoto T., Sasaki E., Matoska V., Bauer P.O, & Oyama F. (2019). High expression of acidic chitinase and chitin digestibility in the stomach of common marmoset (Callithrix jacchus), an insectivorous nonhuman primate. Scientific Reports, 9, 159.

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Affinity Purification of Anti-Phospho-DGLα Antibody

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Polyclonal rabbit anti-serum to a Ser798-phosphorylated (pS798) DGLα was generated by Cocalico Biologicals. Rabbits were immunized with a phosphorylated peptide spanning residues 793-803 of human DGLα (CDSRRSpSGFRSI) following conjugation to Keyhole limpet, and boosted twice after 14 and 21 days. A test bleed (day 35) was screened by immunoblotting lysates of HEK293T cells expressing human V5-DGLα that had been incubated with FSK+IBMX (see Fig. 3). Serum was then collected for affinity purification of antibodies using Sulfo-Link (Thermo) resin coupled to either the non-phosphorylated or pS798 peptides. Serum was first incubated (4°C, 4 h) with non-phosphorylated peptide resin. The unbound material was collected and then incubated with pS798-peptide resin (4°C overnight). After washing the pS798-peptide resin with TBS-Tween (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 0.1% (v/v) Tween-20), antibodies were eluted using 0.2 M glycine pH 2.5, neutralizing the eluate as it was collected in tubes containing Tris-HCl, pH 9.0. Pooled fractions were concentrated using a Centricon-10 device (Millipore-Sigma) and then dialyzed against TBS/50% (v/v) glycerol. Available samples of the pS798-DGLα antibody will be shared upon reasonable request.

Shonesy B.C., Stephenson J.R., Marks C.R, & Colbran R.J. (2020). Cyclic AMP-dependent protein kinase and D1 dopamine receptors regulate diacylglycerol lipase-α and synaptic 2-arachidonoyl glycerol signaling. Journal of neurochemistry, 153(3), 334-345.

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Antibody Production against TMEM30A and ATP8A1

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For polyclonal antibody production against TMEM30A and ATP8A1, peptides with amino acid sequence CKYRNSSNTADITI (the C-terminal 13 residues of mouse TMEM30A plus an N-terminal Cys) and CRAYDTTKQRPDEW (the C-terminal 13 residues of human ATP8A1 plus an N-terminal Cys) were synthesized, conjugated with keyhole limpet hemocyanin and injected into Japanese White rabbits (Kitayama Labes). The antisera were affinity purified with the antigen polypeptide immobilized on a coupling gel (SulfoLink; Thermo Fisher Scientific). All experiments were carried out in accordance with the institutional guidelines on animal experimentation and were approved by the Juntendo University Animal Care and Use Committee (approval number: 200069). All efforts were made to minimize suffering and distress of the animals. Intracardiac puncture for the whole blood collection was performed under deep terminal anesthesia with sodium pentobarbital.

Takasugi N., Araya R., Kamikubo Y., Kaneshiro N., Imaoka R., Jin H., Kashiyama T., Hashimoto Y., Kurosawa M., Uehara T., Nukina N, & Sakurai T. (2018). TMEM30A is a candidate interacting partner for the β-carboxyl-terminal fragment of amyloid-β precursor protein in endosomes. PLoS ONE, 13(8), e0200988.

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