Protein G Dynabeads (ThermoFisher) were incubated with anti-STII antibody (GenScript) for 60 minutes, cross-linked for 30 minutes using 20mM dimethyl pimelimidate (ThermoFisher) diluted in 200 mM triethanolamine (Fisher Scientific), quenched with 150mM monoethanolamine (Fisher Scientific), and washed three times with 1× PBS. T cells were lysed in NP40 Cell Lysis Buffer (ThermoFisher) supplemented with protease and phosphatase inhibitors. Lysates were incubated on ice for 15 minutes and then centrifuged at 10,000×g and 4°C for 10 minutes. Immunoprecipitations were performed according to manufacturer’s instructions where Dynabeads were incubated with equal masses of cleared lysates for 90 minutes at room temperature.
Dimethyl pimelimidate
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Dimethyl pimelimidate is a chemical reagent used for cross-linking proteins in biochemical and molecular biology applications. It is a bifunctional imidoester that forms covalent bonds between primary amine groups, facilitating the study of protein-protein interactions and structural analysis.
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Lab products found in correlation
Microspin column c18 silica, by Nest Group (6 mentions)
Triethanolamine, by Thermo Fisher Scientific (2 mentions)
Monoethanolamine, by Thermo Fisher Scientific (2 mentions)
Protein g dynabead, by Thermo Fisher Scientific (2 mentions)
Anti stii antibody, by GenScript (2 mentions)
Oligo r3 reverse phase resin, by Thermo Fisher Scientific (2 mentions)
Ptmscan ubiquitin remnant motif kit, by Cell Signaling Technology (2 mentions)
Lc ms grade water, by Thermo Fisher Scientific (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Np40 cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
Trifluoroacetic acid, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Sodium deoxycholate, by Merck Group (1 mentions)
Precast polyacrylamide gel, by Bio-Rad (1 mentions)
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Octyl β d glucopyranoside, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic 100x, by Thermo Fisher Scientific (1 mentions)
Blasticidin s hcl, by Thermo Fisher Scientific (1 mentions)
14 protocols using dimethyl pimelimidate
1
Protein G Dynabead Immunoprecipitation Protocol
2
Protein A Magnetic Bead Immunoprecipitation
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PureProteome Protein A magnetic beads were obtained from EMD Millipore (LSKMAGA10). The RPMI-1640 media with L-glutamine, 1X PBS, L-glutamine, fetal bovine serum (FBS) and Antibiotic-Antimycotic (100X) were procured from Gibco (ThermoFisher Scientific). Several lab chemicals including brefeldin A, polybrene, phenylmethylsulfonyl fluoride (PMSF), sodium deoxycholate, octyl-beta-D glucopyranoside, iodoacetamide, triton X-100, triethanolamine (TEA), tris-base, glycine, protease inhibitor cocktail (catalog # P8340) and sodium azide were obtained from Sigma Aldrich. Dimethyl pimelimidate (DMP, catalog # 21667), FITC, acetonitrile, blasticidin S-HCl and trifluoroacetic acid were purchased from ThermoFisher Scientific. Bio-Rad 4–20% precast polyacrylamide gels (catalog # 4561096) were used for SDS-PAGE wherever manual gels were not used. Accugene 0.5 M EDTA solution was obtained from Lonza Bioscience.
3
Antibody Immobilization on Magnetic Beads
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Monoclonal antibodies 6E10, 4G8 and 1G6; msIgG; and polyclonal antibody CT were covalently linked to Protein G-coupled magnetic beads (Dynabeads, Life Technologies; 200 μg of antibody per 1 mL of Dynabead slurry) using dimethyl pimelimidate (Thermo Fisher Scientific) as the crosslinker, according to the manufacturer’s instructions. Prior to use, beads were pre-treated to wash off any antibody that was not covalently linked. Specifically, beads were washed for 15 min at 4 oC with IP Buffer A, then 15 min at 4 oC with IP Buffer B, followed by 5 min at 4 oC with IP Buffer B with 1% (v/v) Trtion X-100. To monitor any shedding of antibody from the beads, the beads were incubated and mixed in 30 μL of elution buffer (100 mM glycine-HCl, 1% (w/v) n-Octyl β-D-thioglucopyranoside (OTG), pH 2.8) for 3 min at room temperature. The eluate was collected, and 10 μL of SDS-PAGE loading buffer was added to the eluate. The sample was boiled at 95°C for 5 min, and loaded onto a 10–20% Tris-Tricine precast gel. Gel electrophoresis and WB were performed as described above, except that goat-anti-msIgG-conjugated to HRP (Jackson ImmunoResearch Laboratories Inc.) or goat-anti-rbt IgG-conjugated to HRP (for CT) (Jackson ImmunoResearch Laboratories Inc.) was used to probe blots.
4
Immobilization and Validation of Tau-13 Antibody
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The tau-13 antibody was immobilized as previously described52 (link). Briefly, the antibody was covalently linked to Protein G magnetic beads (Thermo Fisher Scientific; 200 µg of antibody per 1 mL of bead slurry) using dimethyl pimelimidate (Thermo Fisher Scientific) as the crosslinker. Prior to use, beads were pre-treated with IP buffers A and B to wash off any antibody not covalently linked to beads. To monitor any shedding of antibody from the beads, the beads were treated in elution buffer (100 mM glycine-HCl (pH 2.8) and 1% (w/v) n-Octyl β-D-thioglucopyranoside (OTG)). The eluate was collected and analyzed by WB using HRP-conjugated goat-anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA).
5
Immunoprecipitation of MET Receptor
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Total cell proteins were obtained by lysis with cold RIPA buffer47 (link) in the presence of 1 mM Na3VO4 and a cocktail of protease inhibitors (all from Sigma-Aldrich). 250 µg of total protein lysates were incubated at 4 °C for 2 h on rotor with anti-human MET DO-24 mAb48 (link) covalently conjugated to Sepharose-protein A (GE Healthcare, Buckinghamshire, UK) with Dimethylpimelimidate (Thermo Fisher-Scientific) following standard methods. As control, an equal amount of total proteins was incubated with Sepharose protein A. After five washes with RIPA buffer, immunoprecipitated proteins were eluted with boiling LB and analysed by western blotting.
6
Purification and Antibody Generation of Arl15 Protein
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DNA plasmid, His-Arl15-WT in pET30ax, was used to transform BL21 E coli cells. After induction by isopropyl β-D-1-thiogalactopyranoside, the bacterial cell pellet was lysed by sonication in PBS containing 8 M urea. After high-speed centrifugation, the supernatant was incubated with nickel-nitrilotriacetic acid agarose beads at room temperature for 2 hr. The beads were subsequently washed in PBS containing 8 M urea and 20 mM imidazole. Next, bead-bound His-Arl15-WT was eluted in PBS containing 8 M urea and 250 mM imidazole. After concentrating and changing the buffer to PBS containing 4 M urea, His-Arl15-WT was sent to Genemed Synthesis Inc for rabbit immunization and antiserum collection. To purify the polyclonal antibody against Arl15, GST-Arl15 immobilized on glutathione Sepharose beads was incubated with 50 mM dimethyl pimelimidate (Thermo Fisher Scientific) in 200 mM sodium borate pH 9.0 to crosslink GST-Arl15 covalently onto glutathione Sepharose beads. The crosslinked beads were subsequently washed with 200 mM ethanolamine pH 8.0, incubated with the antiserum at room temperature for 1 hr, and washed with PBS. Finally, the bound antibody was eluted by 100 mM glycine pH 2.8 and dialyzed against PBS.
7
Protein Crosslinking and Analysis
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Efm2 and N‐terminal truncation mutants, in 25 mm sodium phosphate buffer, 100 mm NaCl, 20% (v/v) glycerol, 5 mm β‐mercaptoethanol, 0.2 m triethanolamine, were crosslinked with 330 μm dimethyl pimelimidate (Thermo Fisher Scientific) for 2 h at room temperature. For the 60‐min time course assay of Efm2 crosslinking, 410 μm dimethyl pimelimidate was added instead. SDS was added to a final concentration of 1% prior to addition of the crosslinker for a negative control. SDS sample buffer was added to quench the crosslinking reaction and samples were analysed by SDS/PAGE and immunoblotting as described above.
8
Selenoprotein P Immunoprecipitation and Western Blot
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Preparation of the whole-cell extracts and Western blot analysis were conducted as described previously (32 (link), 33 (link)). Immunoprecipitation assays were conducted using the rat anti-human selenoprotein P (SeP) monoclonal Abs (mAbs), BD1 and BD3 (34 ). BD1 mAb (2 μg) was coupled to Dynabeads Protein G (20 μl, Invitrogen Life Technologies, Carlsbad, CA) using a chemical cross-linker, dimethyl pimelimidate (Thermo Fisher Scientific Inc., Waltham, MA). The BD1 mAb-conjugated Dynabeads (20 μl) were then applied to human serum (1 μl) and incubated for 1 h at 4°C. The beads were washed, and eluted protein samples were subjected to Western blot analysis, as described below. For Western blot analysis, rat anti-human SeP mAb [BD3 (34 ), 1 μg/ml], chicken anti-human extracellular GPx (eGPx) Ab [(35 (link)), 5 μg/ml], and rabbit anti-cellular GPx (cGPx) Ab (1 μg/ml, LF-PA0019; Lab Frontier Co. Ltd., Seoul, Korea) were used. As a loading control for the serum samples, separated proteins were stained with Coomassie Brilliant Blue R-250 (CBB). The major band derived from each serum sample is indicated in Fig. 2
9
Ubiquitin Remnant Motif Enrichment
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A Cell Signaling PTMScan ubiquitin remnant motif kit (Cat#: 5562) was used to enrich for peptides that had been ubiquitinated. Aliquoted beads were cross-linked for 30 minutes in 100 mM sodium borate and 20 mM dimethyl pimelimidate (Thermo Scientific), following the protocol outlined by Udeshi et al.28 (link). Tryptic peptides were resuspended in IAP buffer (50 mM MOPS, pH 7.2, 10 mM sodium phosphate, 50 mM NaCl) and immunoprecipitated with the provided antibody for 2 h at 4°C. Samples were eluted in LC-MS grade water (Thermo Fisher) with 0.15% v/v TFA and separated into either 3 high-pH fractions (enriched ubiquitinated peptides) or 7 high-pH fractions (global proteome) over C18 columns (The Nest Group, MicroSpin column C18 silica, part#: SEM SS18V, lot#: 091317). Fractionated samples were desalted a final time over Oligo R3 reverse-phase resin (Thermo Scientific, Cat#:1-1339-03).
10
Ubiquitin Remnant Motif Enrichment
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