For western blotting, cells were lysed in RIPA buffer (Boston BioProducts) supplemented with protease (Roche) and phosphatase (Roche) inhibitor. Proteins were separated on NuPAGE 4–12% Bis-Tris gradient gels (Invitrogen), transferred to polyvinylidine difluoride membranes (Immobilon P, Millipore) and the blots were probed with the indicated antibodies. For immunoprecipitation, U2OS, DU145, PC3, 293T, and MEF cells were transfected with the indicated expression vectors by using LIPOFECTAMIN 2000 (Life Technologies). Twenty-four hours after transfection, cells were lysed in RIPA buffer with protease (Roche) and phosphatase (Roche) inhibitor. Of total lysates, 500 mg were precleared for 30 minutes at 4°C and then immunoprecipitated with anti-Myc (Cell Signaling Technology 9B11, 1:500), or anti-PTEN (Cell Signaling Technology 9559, 1:500) antibody overnight at 4°C. The Protein-A or Protein-G sepharose beads (GE Healthcare) were then added and incubated for another 2 hours. The immunoprecipitates were washed with RIPA buffer three times. In denaturing conditions, standard Laemmli-Buffer with 5% final concentration of β-mercaptoethanol was added to the samples, which were then boiled and separated on NuPAGE 4–12% Bis-Tris gradient gels (Invitrogen).
Protein a
Manufactured by GE Healthcare
Sourced in United States
Protein A is a laboratory equipment used for the purification of antibodies. It is a genetically engineered protein that binds to the Fc region of immunoglobulins, allowing for the selective capture and separation of antibodies from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Igg elution buffer, by Thermo Fisher Scientific (6 mentions)
Expi293 cell, by Thermo Fisher Scientific (4 mentions)
Protein g sepharose bead, by GE Healthcare (3 mentions)
Protein g, by GE Healthcare (3 mentions)
Anti flag m2 affinity resin, by Merck Group (2 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence reagent, by PerkinElmer (2 mentions)
Protein g sepharose 4 fast flow, by GE Healthcare (2 mentions)
Expi293f cells, by Thermo Fisher Scientific (2 mentions)
Protein g agarose beads, by GE Healthcare (2 mentions)
Immobilon p, by Merck Group (1 mentions)
Nupage 4 12 bis tris gradient gel, by Thermo Fisher Scientific (1 mentions)
Lipofectamin 2000, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Roche (1 mentions)
Anti myc, by Cell Signaling Technology (1 mentions)
Anti pten, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Boston BioProducts (1 mentions)
Protease, by Roche (1 mentions)
Ni nta bead, by Qiagen (1 mentions)
Unicorn 5, by GE Healthcare (1 mentions)
47 protocols using protein a
1
Western Blotting and Immunoprecipitation Methods
2
Recombinant Protein Purification and Characterization
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The recombinant proteins were expressed in Lenti-X 293T cells by calcium phosphate transfection and purified by affinity chromatography with protein A (GE Healthcare) and Ni-NTA beads (Qiagen). Gel filtration of the proteins were performed on an ÄKTA purifier system with Unicorn 5.1 software (GE Healthcare) using PBS as running buffer at a constant flow rate of 0.1 ml/min. 200 μg protein was loaded on a Superdex 200 10/300 GL column (GE Healthcare). Ovalbumin and aldolase of the Gel Filtration HMW Calibration Kit (GE Healthcare) were used as molecular weight controls. Quantifications were done by capillary electrophoresis (Experion Pro260 kit; Bio-Rad). Purity and molecular masses of TP15-Fc and 4D5-Fc (5 μg protein each) were analyzed on Coomassie gel by using standard procedures.
3
Isolation and Purification of VEGFR2-Binding Fab
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KD035 was identified by panning of Dyax FAB‐310 phagemid library on immobilized human VEGFR2 (R&D systems) followed by affinity maturation using a light chain shuffling method. The constructs carrying KD035 antigen‐binding fragment (Fab) domain were cloned into the mammalian expression vector pBh1 (Dyax) and transiently expressed in human 293 Expi cells (Invitrogen). Fabs were purified from cell culture supernatant by passing it several times through a protein A Sepharose HP column (GE Healthcare) using a peristaltic pump for a period of 8 h and eluting with 1 M Glycine pH 2.0. The eluate was further purified by size exclusion chromatography (SEC) using a Superdex 75 increase (GE Healthcare) column.
The variable domain amino acid sequence of IMC‐1121B (which shares the amino acid sequence of ramucirumab as described in US 8057791 B2/EP 1916001 A2) was reverse‐translated into DNA sequences and optimized by using Integrated DNA Technologies tools. The synthesized genes of 1121 light chain and heavy chain variable domains were cloned into a mammalian expression vector pBh1 (Dyax). The plasmid DNA containing 1121 gene was transfected and expressed by using the FreeStyle™ MAX 293 Expression System (ThermoFisher Scientific) according to the manufacture instruction. The supernatant was harvested and purified by protein A (GE) with at least 95% monomer detected by SEC‐UPLC (Waters).
The variable domain amino acid sequence of IMC‐1121B (which shares the amino acid sequence of ramucirumab as described in US 8057791 B2/EP 1916001 A2) was reverse‐translated into DNA sequences and optimized by using Integrated DNA Technologies tools. The synthesized genes of 1121 light chain and heavy chain variable domains were cloned into a mammalian expression vector pBh1 (Dyax). The plasmid DNA containing 1121 gene was transfected and expressed by using the FreeStyle™ MAX 293 Expression System (ThermoFisher Scientific) according to the manufacture instruction. The supernatant was harvested and purified by protein A (GE) with at least 95% monomer detected by SEC‐UPLC (Waters).
4
F240 Fab Preparation and Purification
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F240 was generated as previously described3 (link) using molecular clones of the heavy and light chains provided by Dr. Yongjun Guan. F240 Fabs were prepared from purified IgG (10 mg/ml) by proteolytic digestion with immobilized papain (Pierce, Rockford, IL) and purified using protein A (GE Healthcare, Piscataway, NJ), followed by gel filtration chromatography on a Superdex 200 16/60 column (GE Healthcare, Piscataway, NJ).
5
Transient Fab Fusion Protein Expression
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Example 5
Fab fusion proteins were expressed in a transient mammalian expression system employing pcDNA and HEK293F suspension cell (Invitrogen). The expression constructs were transfected into HEK293F cells (Invitrogen) adding preformed DNA and 25 kD polyethylenimine (PEI) complex (DNA to linear 25 kD PEI at 1:3 ratio by weight) in 1/10 of cell culture volume of F-17 synthetic medium (Invitrogen). Transfected cells grown in 5% moisturized CO2 incubator with shaking were fed with 25 ml of 20% TN1 (Organotechnie SA, France) 24 hr post-transfection. Culture supernatants were usually harvested 5 days post-transfection and proteins were purified using affinity chromatography by either protein A (for human IgG and Fc proteins), protein G (for mouse IgG), or HISTRAP® columns (for his-tagged Fab and his-tagged Fab fusions) (GE Healthcare). After buffer exchange using 10 kD MW cut off spin tubes, proteins were stored in PBS buffer at 4° C. Proteins were usually analyzed by 4-20% SDS-PAGE (polyacrylamide gel electrophoresis, NOVEX® mini gel) under non-reducing and/or reducing condition (5% mercaptoethanol).
6
Isolation of HLA Peptide Complexes
7
Enzyme-Linked Immunosorbent Assay for T-DM1
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N2’-Deacetyl-N2’-(3-mercapto-1-oxopropyl)-maytansine (Mertansine, DM1 compound) was obtained from Abcam (Cambridge, UK). Freund’s complete adjuvant (FCA) and Freund’s incomplete adjuvant (FIA) were obtained from Sigma–Aldrich (St. Louis, MO, USA). BSA-conjugated DM1 was prepared by Abfrontier (Seoul, Republic of Korea). Protein A and Protein G were purchased from GE Healthcare. T-DM1 (trade name Kadcyla®) was purchased from Dongwon Pharmaceutical (Daejeon, Republic of Korea) for laboratory research use. Human anti-trastuzumab, a positive control antibody, was purchased from Bio-Rad (Puchheim, Germany). HER2-ECD was obtained from Sino Biological Inc. (Beijing, China). Human anti-Fc-specific antibody conjugated to peroxidase was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Biotin-conjugated Kadcyla and HER2-ECD were prepared by AbFrontier (Seoul, Republic of Korea). Streptavidin-HRP was purchased from BD PharmingenTM (San Diego, CA, USA). Carbonate-bicarbonate buffer, TMB substrate, and stop buffer were purchased from Sigma–Aldrich (St. Louis, MO, USA). A 1x PBS-T was purchased from Fluka (Buchs, Switzerland). Individual Crl:CD (SD) rat blank sera were obtained from BioChemed (Winchester, VA, USA).
8
Engineered scFv-Fc Antibody Expression
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The scFv regions of KT112 (phage clone) were cloned in‐frame into an expression vector with a signal peptide and the human IgG4 Fc region (S228P/S235E/R409K).17 Immunoglobulin G4 (S228P/S235E/R409K) is a low‐effector‐activity Ab format that shows increased stability. The VH and VL regions of KT112, AM1 (AbbVie), and DNP (negative control Ab) were each cloned in‐frame into an expression vector with a signal peptide and human IgG4 constant region (S228P/S235E/R409K). Similarly, cetuximab (DB00002; DrugBank) was cloned into an expression vector with a signal peptide and human IgG1 constant region. The plasmids were transfected into Expi293F cells using the Expi293 Expression System (Thermo Fisher Scientific). Antibodies and VH‐linker‐VL‐hinge‐CH2‐CH3 (scFv‐Fc) were purified using Protein A (GE Healthcare). After elution, the buffer in which the sample was dissolved was replaced with PBS.
9
Recombinant Plasmodium TatD DNase Proteins
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Genes encoding the TatD-like DNase of P. falciparum, P. berghei and P. chabaudi were cloned from the corresponding parasites using the rtPCR method. The sequences coding for the H303 and D352 mutations in PbTatD were chemically synthesized, and all genes were cloned into the pET-28a and pGEX-4T-1 vectors, respectively (Invitrogen). HIS-tagged and GST-tagged recombinant proteins were expressed in BL21(DE3) cells using the pET-28a and pGEX-4T-1 vectors, respectively, and the soluble proteins were purified using His GraviTrap and Glutathione Sepharose (GE Healthcare), respectively, according to the manufacturer's instructions. Anti-rPfTatD/rPbTatD/rPcTatD sera were raised in New Zealand white rabbits by four immunizations with 300 μg of recombinant Protein And Freund's adjuvant. The sera were collected following titre evaluation. Specific IgGs were purified from the sera using Protein A (GE Healthcare) according to the manufacturer's instructions.
10
ChIP-qPCR for Histone Modifications
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Chromatin immunoprecipitation (ChIP) was carried out as previously described (32 (link)) with the oligonucleotides listed in Supplementary Table S2 . Thus, anti-H3 (1.0 μl; Abcam, 1791), anti-acetyl H4 (1.0 μl; Millipore, 06–598), anti-acetyl H3 (1.0 μl; Millipore, 06-599), or anti-trimethyl H3K4 (0.5 μl; Millipore, 07-473) was bound to Protein A (GE-Healthcare, 17078001) agarose beads. Binding for anti-acetyl H4, anti-acetyl H3 and anti-H3K4me3 or for anti-H3 was done overnight in FA lysis buffer containing 1 M NaCl or in FA lysis buffer with 275 mM NaCl, respectively. Precipitates were washed with the same buffer, once with FA lysis buffer containing 1.5 M NaCl for anti-acetyl H4, anti-acetyl H3 and anti-H3K4me3 or with FA lysis buffer containing 500 mM NaCl for anti-H3, once with 10 mM Tris–HCl (pH 8.0), 0.25 M LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate, and once with TE (10 mM Tris–HCl [pH 8.0], 1 mM EDTA). Precipitated DNAs were analyzed by real-time quantitative PCR using SYBR qPCR Master mix (TOYOBO, QPS-201T) and CFX96 cycler (Bio-Rad).
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