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Aminolink resin

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AminoLink resin is a versatile cross-linked agarose-based material used for the immobilization of proteins, peptides, and other ligands. It provides a stable and controlled method for covalent attachment, enabling efficient affinity purification and other protein-based applications.

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Lab products found in correlation

Pierce direct ip kit, by Thermo Fisher Scientific (2 mentions) Cnbr activated sepharose, by GE Healthcare (2 mentions) Peptide n glycosidase f, by New England Biolabs (2 mentions) Gblock, by Integrated DNA Technologies (1 mentions) Anti flag m2 magnetic bead, by Merck Group (1 mentions) Anti brd4 a301 985a, by Fortis Life Sciences (1 mentions) Anti flag m2 conjugated agarose beads, by Merck Group (1 mentions) Anti α tubulin dm1α, by Merck Group (1 mentions) Anti dsred 632496, by Takara Bio (1 mentions) Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Ni nta resin, by Qiagen (1 mentions) Edta free protease inhibitor, by Roche (1 mentions) Mini protean tgx precast gel, by Bio-Rad (1 mentions) Odyssey nitrocellulose membrane, by LI COR (1 mentions) Anti α tubulin, by Active Motif (1 mentions) Anti histone h3, by Active Motif (1 mentions) Titremax gold adjuvant, by Merck Group (1 mentions) Sodium azide, by Merck Group (1 mentions) Anti flag m2 antibody, by Merck Group (1 mentions) Denaturing buffer 10, by New England Biolabs (1 mentions)

14 protocols using aminolink resin

1

Borrelia burgdorferi Protein Co-IP

Cited 1 time
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Co-IP experiments were performed using the Pierce Direct IP Kit (Thermo Scientific, Rockford, IL). Briefly, B. burgdorferi B31 cells were grown to mid-log phase (5 × 107 organisms ml−1) in complete BSK-II medium before whole-cell lysates were prepared as previously described (Lenhart et al., 2012 (link)). 500 µl of final lysates were then mixed with AminoLink resin with conjugated BB0794, BamA, or GST antibodies following the manufacturer’s instructions (Thermo Scientific). The lysate and resin mixture was rotated end over end at 4°C overnight before being washed. Proteins bound to the resin were eluted by adding 50 µl of final sample buffer containing β-mercaptoethanol and boiled for seven minutes before SDS-PAGE and immunoblot analysis.
Iqbal H., Kenedy M.R., Lybecker M, & Akins D.R. (2016). The TamB ortholog of Borrelia burgdorferi interacts with the β-barrel assembly machine (BAM) complex protein BamA. Molecular microbiology, 102(5), 757-774.
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2

Polyclonal Antibody Production and Validation

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Polyclonal antibodies against stMyHC and MRTF were produced in rabbits (Syd Labs), and polyclonal antibodies against TAGNL2 and TAGNL3 were produced in chickens (Pacific Immunology). The coding region of each antigen (Supplemental Sequences) was cloned into the pET His6 GST TEV LIC cloning vector (Addgene, plasmid #29655) from synthesized gBlocks (Integrated DNA technologies) (stMyHC) or from the E. muelleri cDNA library. Recombinant proteins were expressed in Escherichia coli (Rosetta strain DE3; EMD Millipore) and purified with Pierce Glutathione resin (ThermoFisher Scientific #16101) following the manufacturer’s protocol. Antibodies were affinity purified on a column made by coupling each recombinant protein to AminoLink Resin (ThermoFisher Scientific #20381) and validated by Western blotting and by Peptide Competition Assay (Supplementary Figs. 9–12).
Colgren J, & Nichols S.A. (2022). MRTF specifies a muscle-like contractile module in Porifera. Nature Communications, 13, 4134.
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3

Affinity-Based Interactomics: Mapping Oncogenic Pathways

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We provide a brief summary of publicly available datasets being re-analysed, for improved context of our analysis and results. Briefly, HRAS IP-MS samples were immunoprecipitated (Anti-Flag M2 Magnetic Beads, Sigma) from point mutant baits (via site-directed mutagenesis) and WT baits after using creation of stable cell lines (CAL-33, HET-1A and SCC-25) via lentivirus incorporation17 (link). For KRAS IP-MS cells were transfected with pCEFL-FLAG-KRASWT, or with pCEFL-FLAG-KRASG12C. Cell lysates (with the addition of protease and phosphatase inhibitors) underwent immunoprecipitation (anti-FLAG M2 conjugated agarose beads, Sigma-Aldrich; A2220). For elution and digestion 2 M Urea was used as well as 50 mM Tris-HCl (pH7.5), trypsin and DTT (for digestion).Finally, the BRD4 IP-MS was performed on nuclear extracts using an Anti‐BRD4 (A301‐985 A, Bethyl Labs) antibody (50 µg) was coupled to 100 µl AminoLink resin (Thermo Fisher Scientific).
Kourtis S., Cianferoni D., Serrano L, & Sdelci S. (2024). Detection of differential bait proteoforms through immunoprecipitation-mass spectrometry data analysis. Scientific Data, 11, 551.
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4

Generation of anti-dTAT Antibodies

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The following antibodies were used in this study: anti-acTb (6-B11-1, Sigma), anti-α-tubulin (DM1α, Sigma), FITC-labeled DM1α monoclonal antibodies (Sigma), anti-GFP (A-11122, Fisher), anti-DsRed (632496, Clontech), Anti-Myc (9E11, DSHB), Anti-Futsch (22C10, DSHB), Cy5-conjugated anti-HRP (Jackson immunoresearch), and Alexa Fluor conjugated secondary antibodies (Fisher). In order to generate antibodies against dTAT, we used PCR to amplify the catalytic domain (residues 1 to 196) and cloned this fragment into the NheI/XbaI sites of pET28a or the BamHI/NotI sites of pGEX6P2. Recombinant dTAT 1-196 was expressed in E. coli and purifiied on NiNTA resin (QIAGEN) and glutathione-Sepharose, respectively. Purified 6xhis-dTAT1-196 protein was used to generate polyclonal antibodies in rabbits (Pocono Rabbit Farm) and the antibodies were further affinity-purified on GST-dTAT 1-196 bound to amino-link resin (Thermo Fisher Scientific). Secondary antibodies for immunofluorescence were purchased from Jackson Immunoresearch. HRP-conjugated secondary antibodies for immunoblots were purchased from Sigma.
Yan C., Wang F., Peng Y., Williams C.R., Jenkins B., Wildonger J., Kim H.J., Perr J.B., Vaughan J.C., Kern M.E., Falvo M.R., O’Brien ET I.I.I., Superfine R., Tuthill J.C., Xiang Y., Rogers S.L, & Parrish J.Z. (2018). Microtubule Acetylation Is Required for Mechanosensation in Drosophila. Cell reports, 25(4), 1051-1065.e6.
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5

Antibody Production and Purification

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Antibodies against MSL1[423-1030], MSL2[421-540], CLAMP and MSL3 were raised in rabbits and purified from the sera by ammonium sulfate fractionation followed by affinity purification on CNBr-activated Sepharose (GE Healthcare, USA) or Aminolink Resin (ThermoFisher Scientific, USA) according to standard protocols. Other antibodies: mouse monoclonal anti-FLAG, clone M2 (F1804, Sigma, USA), mouse monoclonal anti-HA, clone HA-7 (H3663, Sigma, USA), mouse monoclonal anti-lamin Dm0, clone ADL84.12 (ADL84.12, DSHB, USA).
Tikhonova E., Revel-Muroz A., Georgiev P, & Maksimenko O. (2024). Interaction of MLE with CLAMP zinc finger is involved in proper MSL proteins binding to chromosomes in Drosophila. Open Biology, 14(3), 230270.
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6

Borrelia burgdorferi Protein Co-IP

Cited 1 time
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Co-IP experiments were performed using the Pierce Direct IP Kit (Thermo Scientific, Rockford, IL). Briefly, B. burgdorferi B31 cells were grown to mid-log phase (5 × 107 organisms ml−1) in complete BSK-II medium before whole-cell lysates were prepared as previously described (Lenhart et al., 2012 (link)). 500 µl of final lysates were then mixed with AminoLink resin with conjugated BB0794, BamA, or GST antibodies following the manufacturer’s instructions (Thermo Scientific). The lysate and resin mixture was rotated end over end at 4°C overnight before being washed. Proteins bound to the resin were eluted by adding 50 µl of final sample buffer containing β-mercaptoethanol and boiled for seven minutes before SDS-PAGE and immunoblot analysis.
Iqbal H., Kenedy M.R., Lybecker M, & Akins D.R. (2016). The TamB ortholog of Borrelia burgdorferi interacts with the β-barrel assembly machine (BAM) complex protein BamA. Molecular microbiology, 102(5), 757-774.
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7

Immunoprecipitation of Mitochondrial Proteins

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Cells were lysed with RIPA buffer containing EDTA-free protease inhibitors (Roche) and 25 units alkaline phosphatase (NEB) per ml cell lysate. After centrifugation, supernatants were incubated with anti-MFN1, -MFN2 (Origene), -OPA1 (cell signaling) or -WBSCR16 (Abcam) antibodies coupled to Aminolink Resin (Pierce Direct IP kit, Thermo Scientific). Resin/antibody-bound proteins were subjected to SDS-PAGE and immunoblotting. Additional details are in Supplemental Experimental Procedures, as is detailed description of in vitro pull down assays of recombinant proteins.
Huang G., Massoudi D., Muir A.M., Joshi D.C., Zhang C.L., Chiu S.Y, & Greenspan D.S. (2017). WBSCR16 is a Guanine Nucleotide Exchange Factor (GEF) Important for Mitochondrial Fusion. Cell reports, 20(4), 923-934.
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8

Purification and Characterization of Peanut Allergens

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A chromatographic fraction of 2S albumins from raw peanuts was obtained as previously described [15 (link)], which consists of >97% Ara h 2 (79 %) and Ara h 6 (18 %). Other allergen detected (<3% by weight) are Ara h 7 and Ara h 8. Total IgE was affinity purified from peanut-allergic sera using affinity-purified goat anti-human IgE [49 (link)] coupled to AminoLink® resin (ThermoFisher, Waltham, MA, USA). IgE that bound to either Ara h 2 or Ara h 6 (Ara h 2/6-IgE) was then purified using Ara h 2/6 coupled to AminoLink® resin. Purification of Ara h 2 and Ara h 6 from raw peanut was as previously described [16 (link)]. Four individual rabbits were each immunized at monthly intervals five times SQ with 500μg either purified Ara h 2 (2 rabbits) or purified Ara h 6 (2 rabbits) with a mixture of complete Freund/incomplete Freund's adjuvant (YenZym, Inc., South San Francisco, CA). IgG from all sera were demonstrated to recognize the purified allergens in an ELISA assay.
Chen X., Negi S.S., Liao S., Gao V., Braun W, & Dreskin S.C. (2016). Conformational IgE epitopes of peanut allergens Ara h 2 and Ara h 6. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 46(8), 1120-1128.
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9

Heterodimer Detection in Anopheles Immunity

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Sf9 cells were infected at MOI of 0.1 and conditioned media collected 96 hpi. For non-reducing SDS-PAGE DTT was excluded from the 6 × loading buffer; samples were not heated prior to electrophoresis on 4–20% mini protean TGX precast gels (BioRad). Gels were transferred to Odyssey nitrocellulose membrane (LI-COR) at 30 V overnight in Towbin buffer with 10% methanol; but 1% SDS in the electrophoresis buffer was necessary for efficient transfer of the heterodimer in non-reducing conditions. Western blotting was performed with anti-6 × His mouse mAb (Clontech), with secondary IRDye 800CW goat anti-mouse (LI-COR) for. An. gambiae CTL4.
For detection of CTLMA2, full length His-tagged CTLMA2 was expressed in sf9 cells and purified from conditioned media by Co (Talon) affinity chromatography. The protein was used for inoculation into a rat for production of polyclonal antibodys by Cocalico Biologicals (Stevens, PA). The resulting antiserum was affinity purified using recombinant CTLMA2 immobilized on Aminolink resin (Thermo Fisher). Western blotting was performed with affinity purified anti-CTLMA2, with secondary IRDye 680LT goat anti-rat (LI-COR).
Bishnoi R., Sousa G.L., Contet A., Day C.J., Hou C.F., Profitt L.A., Singla D., Jennings M.P., Valentine A.M., Povelones M, & Baxter R.H. (2019). Solution structure, glycan specificity and of phenol oxidase inhibitory activity of Anopheles C-type lectins CTL4 and CTLMA2. Scientific Reports, 9, 15191.
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10

Antibodies for Drosophila CTCF Characterization

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Antibodies against the N-terminal domain (anti-dCTCF_N) of dCTCF (amino acids 1–287) and amino acids 657–818 of the C-terminal domain (anti-dCTCF_C) were produced in rabbits and purified by affinity purification on Aminolink resin (Thermo Fisher Scientific, USA) according to standard protocols. These antibodies were characterized in a previous study [34 (link)]. The other antibodies used were as follows: mouse monoclonal anti-HA antibodies, clone HA-7 (#H3663, Sigma, USA); mouse monoclonal anti-FLAG antibodies, clone M2 (F1804, Sigma, USA); mouse monoclonal anti-lamin Dm0, clone ADL84.12 (#ADL84.12, DSHB, USA); rat anti-CP190; anti-histone H3 (#39163, Active Motif, USA); anti-α-tubulin (#39527, Active Motif, USA).
Kamalyan S., Kyrchanova O., Klimenko N., Babosha V., Vasileva Y., Belova E., Fursenko D., Maksimenko O, & Georgiev P. (2024). The N-terminal dimerization domains of human and Drosophila CTCF have similar functionality. Epigenetics & Chromatin, 17, 9.
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