Bio-Rad protein assay dye reagent [50 (link)], iProof DNA polymerase, NuviaTM IMAC resin, Precision plus protein dual color standards, and reagents for protein electrophoresis were mainly purchased from Bio-Rad, Hercules, CA, USA. Restriction endonucleases, PrimeSTAR DNA polymerase, and the In-Fusion HD cloning kit were obtained from Takara, Japan. SeaKem® LE Agarose was purchased from Lonza, Morristown, NJ, USA. The PrestoTM Mini Plasmid kit and GenepHlowTM Gel/PCR kit were supplied by Geneaid, New Taipei City, Taiwan. Goat anti-mouse IgG H&L (HRP, ab6789) and a colorimetric phosphate assay kit—PiColorLockTM (ab27004) were obtained from Abcam, Cambridge, UK. Immobilon PVDF membranes, Western Chemiluminescent HRP substrate (ECL), and anti-6x His tag monoclonal antibodies (Cat. # 05-949, Lot # 2869961) were purchased from Merck Millipore, Burlington, MA, USA. Di-C18:0 PA (Lot # 830865P-25MG-C-045) was obtained from Avanti Polar Lipids, Alabaster, AL, USA. Oligonucleotide synthesis and DNA sequencing were carried out by Tri-I Biotech, New Taipei City, Taiwan. All other chemicals were reagent grade.
Nuvia imac resin
Manufactured by Bio-Rad
Sourced in United States
Nuvia™ IMAC Resin is a versatile immobilized metal affinity chromatography (IMAC) resin designed for the purification of recombinant proteins. It utilizes a porous resin matrix and chelated metal ions to selectively capture and purify target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Restriction endonuclease, by Takara Bio (1 mentions)
Iproof dna polymerase, by Bio-Rad (1 mentions)
Protein assay dye reagent, by Bio-Rad (1 mentions)
Precision plus protein dual color standard, by Bio-Rad (1 mentions)
Primestar dna polymerase, by Takara Bio (1 mentions)
Presto mini plasmid kit, by Geneaid (1 mentions)
Genephlow gel pcr kit, by Geneaid (1 mentions)
Goat anti mouse igg h l hrp ab6789, by Abcam (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Seakem le agarose, by Lonza (1 mentions)
Restriction endonuclease enzymes, by Takara Bio (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Protein assay dye reagent concentrate, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Transient expression system, by Thermo Fisher Scientific (1 mentions)
Spectramax abs plus absorbance microplate reader, by Molecular Devices (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Thermo Fisher Scientific (1 mentions)
Nunc maxisorp 96 well plate, by Thermo Fisher Scientific (1 mentions)
Anti mouse iga, by Southern Biotech (1 mentions)
5 protocols using nuvia imac resin
1
Bacterial Protein Expression and Purification
2
Protein Purification and Molecular Manipulations
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Bio-Rad protein assay dye reagent concentrate [48] (link), Nuvia TM IMAC resin, and general chemicals for protein electrophoresis were purchased from Bio-Rad, Hercules, CA, USA. Restriction endonuclease enzymes, DNA proofreading polymerase, PrimeStar, and T4 DNA ligase for molecular biology manipulations were obtained from Takara, Kusatsu, Shiga, Japan. Plasmid mini-preparation kit and gel extraction/PCR cleanup kit were from Geneaid Biotech, New Taipei City, Taiwan. Oligonucleotide synthesis and DNA sequencing were serviced by Tri-I Biotech, New Taipei City, Taiwan. L-phenylalanine, L-tyrosine,
3
BPIFB4 Protein Purification Protocol
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HEK293T cells were transfected with the WT-BPIFB4 or RV-BPIFB4 vector cloned in fusion with His-Tag. The recombinant protein was purified using affinity Nuvia IMAC Resin (Bio-Rad) under native conditions.
4
SARS-CoV-2 S RBD Antibody Detection
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S protein receptor-binding domain (S RBD)-specific antibody titers in serum and BALF were determined using ELISAs. The recombinant His-tagged SARS-CoV-2 S RBD protein (aa 319–541) was produced in Expi293F cells using a transient expression system (Thermo Fisher) according to the manufacturer's instructions and purified using Nuvia™ IMAC Resin (Bio-Rad). NUNC-MaxiSorp™ 96-well plates (Thermo Scientific) were coated with 1 μg/ml of recombinant SARS-CoV-2 S RBD protein overnight at 4 °C and blocked with 5% fetal bovine serum (FBS) in DBPS for 1 h at RT. Serially diluted samples were added to wells and incubated overnight at 4 °C. After washing four times with ELISA washing buffer (DPBS containing 0.05% Tween-20), horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) or anti-mouse IgA (Southern Biotech, Birmingham, AL, USA) antibodies were used to detect S RBD-specific antibodies in the samples. Plates were incubated for 1 h at RT and washed four times with ELISA washing buffer. TMB (eBioscience, San Diego, CA, USA) was used as the substrate, and relative titers were calculated based on the absorbance at 450 nm using a SpectraMax® ABS Plus Absorbance Microplate Reader (Molecular Devices, San Jose, CA, USA).
5
Site-Specific Antibody Conjugation via Sortase
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An LPETG recognition motif was genetically encoded in the construct of the targeting ligand (αHER2LPETG-IgG) for SrtA to site-specifically cleave and conjugate a click reactive azide moiety (SMAB). The expressed and purified αHER2LPETG-IgG was mixed with SrtA and the bifunctional G-PEG4-Azide linker in a 1:6:3 M ratio, respectively. The reaction proceeded for 5 h at 37 °C in the presence of sortase reaction buffer (50 mM Tris, 150 mM NaCl, 0.5 mM CaCl2 pH 8.0). Finally, purification of αHER2Azide-IgG was completed using a Nuvia™ IMAC Resin (Biorad ltd) as described by manufacturer. The flow-through containing αHER2Azide-IgG only was collected and dialyzed against PBS overnight at 4 °C, as SrtA would be removed from the reaction product due to the his-tag, in its construct, affinity to the resin. BCA assays were performed for colorimetric quantification of SMAB-modified ligand (αHER2Azide-IgG) concentrations.
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