The Regdanvimab and its analogs DNA sequences variable regions were synthesized in GenScript. The heavy and light chain variable regions were inserted into pfuse2ss-chig-hg1 and pfuse2ss-clig-hl2 (InvivoGen) vector accordingly. Antibodies were produced using ExpiCHO cells (Thermo Fisher). 8 days after transfection, antibodies were purified from medium using Protein A/G resin (Thermo Fisher). After dialysis in PBS, antibodies were used in neutralization assay. Antibodies’ concentrations were measured by BCA (Bicinchoninic Acid) Protein Assay (Thermo Fisher).
Protein a g resin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Protein A/G resin is a chromatography media designed for the purification of antibodies. It consists of recombinant protein A and protein G immobilized on agarose beads. The resin is used to capture and purify immunoglobulins from complex samples, such as cell culture supernatants or tissue extracts.
Automatically generated - may contain errors
Lab products found in correlation
Bicinchoninic acid bca protein assay, by Thermo Fisher Scientific (1 mentions)
Pfuse2ss clig hl2, by InvivoGen (1 mentions)
Expicho cells, by Thermo Fisher Scientific (1 mentions)
Wallac victor 3 multilabel counter, by PerkinElmer (1 mentions)
Renilla substrate, by Promega (1 mentions)
Cfx384 touch machine, by Thermo Fisher Scientific (1 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Igf2bp1 antibody, by Cell Signaling Technology (1 mentions)
Superscript 3 rt, by Thermo Fisher Scientific (1 mentions)
6 protocols using protein a g resin
1
Generation and Characterization of Regdanvimab Antibodies
2
LIPS Assay for Antibody Detection
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LIPS assay (Figure S1) was performed according to a protocol previously described by Burbelo et al. [19 (link)] with modification. Renilla antigen was prepared by transfecting 293FT cells with Renilla luciferase-orf3b expression plasmid, followed by lysis at 48 h post-transfection. Heat-inactivated patient sera were 1:100 diluted and incubated with 1×107 light units of Renilla antigen on a rotary shaker. 1.5 µL of Protein A/G resin (Thermo Fisher Scientific, USA) diluted in PBS was then added to each sample and incubated to capture the antibody–antigen complex. Following three washes, the beads were transferred to an opaque 96-well plate. Renilla substrate (Promega, USA) was then added and luciferase signal was measured using the Wallac Victor3 Multilabel Counter (PerkinElmer, USA).
3
Immunoprecipitation of Epitope-Tagged Proteins
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Yeast cells expressing epitope-tagged proteins were grown to an OD600 of 0.5–0.8. Cells were harvested and spheroplasted with lyticase (Scott and Schekman, 1980 (link)). Spheroplasts were lysed mechanically in the lysis buffer (1× phosphate-buffered saline [PBS], 5 mM EDTA, pH 8.0, with protease inhibitors added) by using a homogenizer (Wheaton, Millville, NJ). Cell lysates were treated with 0.5% Tween-20 and cleared by centrifugation at 13,000 × g for 10 min. Immunoprecipitations were performed by adding anti-GFP antibody (Ferro-Novick lab) to the supernatant at 4°C and incubated over night. The protein A/G resin (Thermo Scientific, Waltham, MA) was added to the samples, which were further incubated for 2 h at 4°C the next morning. After washing of the affinity resin with PBS immunoprecipitation buffer four times, immunoprecipitated proteins were eluted and reduced with sample buffer (6 M urea, 150 mM Tris-HCl, pH 6.8, 6% SDS, 10% β-mercaptoethanol, bromophenol blue) and analyzed by SDS–PAGE and immunoblotting. The intensities of Western blot bands were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).
4
Immunoprecipitation and Pull-down Assays for CENP-E Interactions
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For immunoprecipitation, synchronized HeLa cells were lysed in IP buffer (50 mM Tris-HCl, pH 8.0; 120 mM NaCl; 0.2% NP-40) supplemented with protease inhibitor cocktail. After pre-clearing with protein A/G resin (ThermoFisher Scientific), the lysate was incubated with CENP-E antibody at 4°C for 24 h with gentle rotation. protein A/G resin was then added into the cell lysates to incubate for another 6 h followed by spun down and washed five times with lysis buffer. The immunoprecipitates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.
For pull-down assays, GST-PRC1-bound sepharose beads were incubated with HEK293T cell lysates containing ectopically expressed GFP-tagged CENP-E or with purified His-tagged CENP-E from bacteria in PBS containing 0.2% Triton X-100 at 4°C for 4 h. The binding fraction was washed with PBS for three times and analyzed by Coomassie Brilliant Blue stained SDS-PAGE gel.
For pull-down assays, GST-PRC1-bound sepharose beads were incubated with HEK293T cell lysates containing ectopically expressed GFP-tagged CENP-E or with purified His-tagged CENP-E from bacteria in PBS containing 0.2% Triton X-100 at 4°C for 4 h. The binding fraction was washed with PBS for three times and analyzed by Coomassie Brilliant Blue stained SDS-PAGE gel.
5
RNA-Seq Analysis of Igf2bp1-Bound Small RNAs in Mouse ESCs
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2‐day LIF‐ or RA‐treated mouse ESCs were UV‐crosslinked at 450 mJ/cm2. Cell extracts were made using a lysis buffer (100 mM Tris pH 7.4, 150 mM NaCl, 1% NP40, RNase inhibitor, protease inhibitor cocktail, and 1 mM DTT). 1 mg of cell extract was taken for immunoprecipitation following which extracts were pre‐cleared with 50 μl protein A/G resin (Thermo Scientific, 53132) for an hour at 4°C. Pre‐cleared lysates were incubated with Igf2bp1 antibody (Cell Signaling Technologies, 2852S) or IgG (Cell Signaling Technologies, 3900S) control overnight at 4°C. Antibody was immunoprecipitated using protein A/G‐coated resin and subsequently washed four times with lysis buffer. To release the RNA from the protein complex, the pulldown sample was treated with 5 mg/ml proteinase K at 37°C for 10 min. The RNA was extracted using TRIzol. Small RNA libraries were made using the isolated RNA as described using TruSeq Small RNA Prep Kit. The libraries were sequenced and analyzed as discussed previously. For qPCR analysis of target genes, 1 μl of the suspended RNA was reverse‐transcribed with SuperScript III RT (Invitrogen) using random hexamers or tsRNA‐specific RT primer. qPCR of the reverse‐transcribed cDNA was carried out on Bio‐Rad CFX384 Touch machine using Fermentas Power SYBR Green Master Mix and respective gene‐specific or tsRNA‐specific primers.
6
Quantification of Glomerular Rabbit IgG
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To quantify deposited rabbit IgG in glomeruli by immunoblot analysis, glomeruli-number-adapted lysates were transferred to an appropriate amount of protein A/G resin (static binding capacity of 20 mg IgG per ml, Thermo Fisher, #53135) and incubated over night at 4°C. The samples were centrifuged for 10 min, 14,000 × g, 4°C, the supernatant was discarded. Rabbit IgG was released from the protein G resin by denaturation with an equal amount of 2 x laemmli buffer. Samples were loaded on a SDS-PAGE under denaturing conditions for immunoblotting to rabbit IgG. As the experiments depict the amount of rabbit IgG that was pulled down from the same number of glomeruli, an immunoblot loading control is technically not providable.
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