Sulfolink immobilization kit for peptides

Manufactured by Thermo Fisher Scientific

Sourced in United States

The SulfoLink Immobilization Kit for Peptides is a laboratory product designed for the covalent immobilization of peptides. The kit includes the necessary components to facilitate the immobilization process.

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Lab products found in correlation

Igg elution buffer, by Thermo Fisher Scientific (1 mentions) Peroxidase antibody, by Merck Group (1 mentions) T6074, by Merck Group (1 mentions) Hitrap protein g hp, by GE Healthcare (1 mentions) Hitrap protein g hp column, by GE Healthcare (1 mentions) Rpmi 1640, by Nacalai Tesque (1 mentions) Ab7671, by Abcam (1 mentions) Ktaprime plus chromatography system, by GE Healthcare (1 mentions) Mouse anti α tubulin dm1a, by Santa Cruz Biotechnology (1 mentions) Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions) Mouse anti myc 9b11, by Cell Signaling Technology (1 mentions) Rabbit anti gst, by Cell Signaling Technology (1 mentions) Ultracel 10 membrane, by Merck Group (1 mentions) Imject maleimide activated mcklh spin kit, by Thermo Fisher Scientific (1 mentions) Imject maleimide activated mcklh, by Thermo Fisher Scientific (1 mentions) Supersignal west femto detection kit, by Thermo Fisher Scientific (1 mentions)

16 protocols using sulfolink immobilization kit for peptides

Generation of Polyclonal Antibodies against C11orf53/OCA-T1 and POU2F3

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Peptides GDPAHFLFRDSWEQTLPD and EADTGSLHDPSPWVKEDGS of C11orf53/OCA-T1 were purchased from GenScript with KLH conjugation. A mixture of both peptides (4 mg each) was used for rabbit immunizations for two rabbits with a 73 day rabbit antibody production protocol from Pocono Rabbit Farm & Laboratory. The whole-blood serum of exsanguination was used to validate the efficiency of antibodies for western blot and immunoprecipitation with HEK293T HA–OCA-T1 overexpression cell lysates. The whole-blood serum was then aliquoted in 10 ml and stored at −80 °C for long-term storage. An aliquot of 10 ml blood serum was then purified with columns containing the mixture of both antigens using the Thermo Fisher Scientific SulfoLink Immobilization Kit for Peptides and eluted 2 ml with IgG elution buffer (Thermo Fisher Scientific, 44999), neutralized with 200 μl 1 M Tris (pH 7.5) and stored at −20 °C with 50% glycerol. This purified antibody was used for western blot and chromatin immunoprecipitation assays.
For POU2F3, the same procedures were followed, except a mixture of peptides NSRPSSPGSGLHASSPTC, ASQNNSKAAMNPSSAAFNC and SSGSWYRWNHPAYLHC was used as antigens for rabbit immunization.

Wu X.S., He X.Y., Ipsaro J.J., Huang Y.H., Preall J.B., Ng D., Shue Y.T., Sage J., Egeblad M., Joshua-Tor L, & Vakoc C.R. (2022). OCA-T1 and OCA-T2 are coactivators of POU2F3 in the tuft cell lineage. Nature, 607(7917), 169-175.

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Affinity-purified MNS3 Antibody Protocol

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The following primary antibodies were used in this study: mouse anti-GFP antibody (11814460001, Roche; 1:2000 dilution); mouse anti-RFP antibody (6G6, Chromotek; 1:2000 dilution); and mouse anti-alpha-tubulin antibody (T6074, Sigma; 1:5000 dilution). The secondary antibodies were as follows: rabbit anti-mouse IgG (whole molecule)–Peroxidase antibody (A9044, Sigma; 1:10,000 dilution); and goat anti-rabbit IgG (whole molecule)–Peroxidase antibody (A0545, Sigma; 1:100,000 dilution). For the generation of MNS3-specific antibodies, a peptide that corresponds to the MNS3 C-terminus was synthesized (LDKFVFNTEAHPLPIRRNT). The MNS3 peptide was coupled to keyhole limpet hemocyanin and then injected into rabbits. The peptides and antisera were prepared by Gramsch Laboratories (Schwabhausen, Germany). Affinity-purified antibodies were prepared using the SulfoLink Immobilization Kit for Peptides (44999, Thermo Scientific). Coupling of the peptides and subsequent affinity purification were performed following the instructions of the manufacturer. The antibody was used at a 1:2000 dilution. Its specificity was tested by immunoblotting studies with recombinant MNS3 produced as described^{7 (link)} and protein extracts of Col-0 and mns1 mns2 mns3^{7 (link)} knockouts (Supplementary Fig. 15).

Schoberer J., König J., Veit C., Vavra U., Liebminger E., Botchway S.W., Altmann F., Kriechbaumer V., Hawes C, & Strasser R. (2019). A signal motif retains Arabidopsis ER-α-mannosidase I in the cis-Golgi and prevents enhanced glycoprotein ERAD. Nature Communications, 10, 3701.

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Synthesis and Purification of Tc_5171 Antibodies

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The immunodominant peptide (VMRIVRALPE) was chemically synthesized and coupled to Keyhole limpet hemocyanin (KLH) using standard methods at the Facility for Biotechnology Resources, CBER, FDA^{52 (link)}. Rabbits were immunized with 125 μg per dose of either the KLH-peptide or the purified recombinant Tc_5171 antigen, with three antigen boosts at 21-day intervals to generate polyclonal sera. Antibodies against the VMRIVRALPE peptide were affinity purified from the serum of the immunized rabbit using the SulfoLink Immobilization Kit for Peptides from Thermo Fisher. Briefly, the VMRIVRALPE peptide, synthesized with an N-terminal cysteine was covalently coupled to the iodoacetyl group on agarose beads. Peptide coupled beads were incubated with 1:2 dilution of rabbit serum in PBS. Peptide bound antibodies were eluted using 0.1 M Glycine HCl and neutralized using 1 M Tris, HCl pH 9.0. Polyclonal IgG antibodies generated against the full-length recombinant Tc_5171 antigen were purified using a HiTrap Protein G HP antibody purification column from GE Lifesciences using manufacturer recommended protocol. Purified antibodies were concentrated and dialyzed in HBS.

Nagarkatti R., Acosta D., Acharyya N., de Araujo F.F., Elói-Santos S.M., Martins-Filho O.A., Teixeira-Carvalho A, & Debrabant A. (2020). A novel Trypanosoma cruzi secreted antigen as a potential biomarker of Chagas disease. Scientific Reports, 10, 19591.

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Affinity Purification of Rabbit Antiserum

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Rabbit antiserum raised against a synthetized peptide encoding the sequence of amino acids 1 to 14 of BmGr9 [NH₂-C-MPPSPDLRADEPKT-COOH ] (Eurofins Genomics) was affinity-purified using the SulfoLink Immobilization Kit for Peptides (Thermo Fisher Scientific).

Morinaga S., Nagata K., Ihara S., Yumita T., Niimura Y., Sato K, & Touhara K. (2022). Structural model for ligand binding and channel opening of an insect gustatory receptor. The Journal of Biological Chemistry, 298(11), 102573.

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Polyclonal Antibody Generation against NHE11

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Polyclonal antiserum against NHE11 was generated in rabbits using a synthetic 12 amino acid peptide corresponding to amino acids 965–976 of the deduced rat NHE11 amino acid sequence. Briefly, in order to produce the NHE11 polyclonal antibody, the unique peptide sequence (NH₂-LLKREIEYTAIC-COOH) was synthesized by the Antibody Research Corporation (Saint Peters, MO, USA), where it was conjugated to keyhole limpet hemocyanin (KLH) and used to immunize rabbits. Our NHE11 peptide (above) was conjugated to a column containing a 6% crosslinked agarose support using a SulfoLink Immobilization Kit for Peptides (ThermoFisher). Sera from the injected rabbits were then affinity-purified using this column. The concentration of purified NHE11 antibody was quantified spectrophotometrically at 280 nm.

Gardner C.C, & James P.F. (2023). The SLC9C2 Gene Product (Na+/H+ Exchanger Isoform 11; NHE11) Is a Testis-Specific Protein Localized to the Head of Mature Mammalian Sperm. International Journal of Molecular Sciences, 24(6), 5329.

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Generating Phospho-Specific PARIS Antibodies

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Antibodies specific to PARIS that is phosphorylated at serine 613 (anti-pS613-PARIS antibodies) were raised by immunizing a rabbit with a phospho-peptide (CDPFK-(phosphorylated)S-PASKGPLASTD, synthesized in the Johns Hopkins Sequencing Facility) and purified in a column conjugated with the phosphorylated PARIS peptide (SulfoLink Immobilization Kit for Peptides, Thermo Scientific).

Lee Y., Stevens D.A., Kang S.U., Jiang H., Lee Y.I., Ko H.S., Scarffe L.A., Umanah G.E., Kang H., Ham S., Kam T.I., Allen K., Brahmachari S., Kim J.W., Neifert S., Yun S.P., Fiesel F.C., Springer W., Dawson V.L., Shin J.H, & Dawson T.M. (2017). PINK1 primes parkin-mediated ubiquitination of PARIS in dopaminergic neuronal survival. Cell reports, 18(4), 918-932.

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Purification of Anti-OR and Anti-SR Antibodies

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To prepare anti-OR and anti-SR antibodies for ADCI experiments, the antibodies were purified from Ugandan high antibody titer serum pool (SE36-positive serum pool) [9] (link). Prior to the purification of antibodies specific to the OR or SR region, whole antibodies were purified from the serum pool with HiTrap Protein G HP columns (GE Healthcare UK Ltd, Buckinghamshire, UK). Antibodies purified by Protein G columns were then loaded to either OR or SR-specific peptide columns. The OR/SR peptide columns were prepared with SulfoLink Immobilization Kit for Peptides (Thermo Fisher Scientific, Waltham, MA), so that OR/SR peptides with cysteine residues at the N-termini (series II peptides 1 and 3, Table S2) were immobilized to the columns via thiol groups, following recommendations of the manufacturer. Antibodies bound to the columns were eluted with 0.1 M Gly-HCl at pH 2.7 and immediately neutralized with 1 M Tris-HCl at pH 8.5.
Murine serum pool from SE36-vaccinated mice was used to purify the antibody against the whole SE36 molecule. However, due to limited sample volume, after applying to Protein G column no further purification was done. All antibodies were dialyzed against RPMI 1640 (Nakalai Tesque, Kyoto, Japan) prior to ADCI assays.

Yagi M., Bang G., Tougan T., Palacpac N.M., Arisue N., Aoshi T., Matsumoto Y., Ishii K.J., Egwang T.G., Druilhe P, & Horii T. (2014). Protective Epitopes of the Plasmodium falciparum SERA5 Malaria Vaccine Reside in Intrinsically Unstructured N-Terminal Repetitive Sequences. PLoS ONE, 9(6), e98460.

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Polyclonal Antibody Generation against MEIOC

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A polyclonal antibody against MEIOC was raised in rabbits against C-terminal peptide CHESINSSNPMNQRGETSKH (YenZym Antibodies, LLC), and affinity purified using the antigenic peptide (SulfoLink Immobilization Kit for Peptides, ThermoScientific).

Soh Y.Q., Mikedis M.M., Kojima M., Godfrey A.K., de Rooij D.G, & Page D.C. (2017). Meioc maintains an extended meiotic prophase I in mice. PLoS Genetics, 13(4), e1006704.

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Generation and Validation of TPC1 and TPC2 Antibodies

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The TPC1 antibody was generated against the N-terminal Cys-peptide corresponding to amino acid region 774–790 of murine TPC1 (C-YQEEIQEWYEEHAREQE). Peptides were obtained from Intavis. The TPC2 antibody was generated by immunizing rabbits with the N-terminal Cys-peptide corresponding to amino acid region 705–726 of murine TPC2 (C-FRDILEEPKEEELMEKLHKHPH). Sera from immunized rabbits were peptide affinity purified employing the SulfoLink Immobilization Kit for Peptides (Thermo Scientific) according to the manufacturer´s protocol and an ÄKTAprime plus chromatography system (GE Healthcare Life Sciences). Antibody elution was performed by applying a linear pH gradient. The TPC1 antibody was first described in Arndt et al. (2014 (link)) and Castonguay et al. (2017 (link)); the TPC2 antibody first in Grimm et al. (2014 (link)).
Anti-alpha 1 sodium potassium ATPase antibody ab7671 was provided from abcam.

Mallmann R.T, & Klugbauer N. (2020). Genetic Inactivation of Two-Pore Channel 1 Impairs Spatial Learning and Memory. Behavior Genetics, 50(6), 401-410.

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Antibody Generation and Characterization

Cited 1 time

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Anti-GCAP1 and anti-GCAP2 polyclonal antibodies have been previously described^{22 (link)}. The polyclonal antibodies against RetGC1 and RD3 were generated in rabbit against C-terminal peptides corresponding to the last 21aa of murine RetGC1 and the last 16aa of murine RD3. Peptides were conjugated to keyhole limpet hemocyanin carrier protein (Imject maleimide activated carrier protein spin kit, Thermo Fisher Scientific) for inoculation in rabbits. A 90-day, three antigen-injection protocol was followed. Serum from immunized rabbits was purified by affinity chromatography by using the immunizing peptide covalently coupled to an agarose column by –SH chemistry (Sulfolink immobilization kit for peptides, Thermo Fisher Scientific). The specificity of the antibodies is shown by Western blot analysis of whole retinal extracts in Supplementary Fig. 1.

Plana-Bonamaisó A., López-Begines S., Andilla J., Fidalgo M.J., Loza-Alvarez P., Estanyol J.M., Villa P.D, & Méndez A. (2020). GCAP neuronal calcium sensor proteins mediate photoreceptor cell death in the rd3 mouse model of LCA12 congenital blindness by involving endoplasmic reticulum stress. Cell Death & Disease, 11(1), 62.

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