FP assays were performed in ES2 buffer (100 mM potassium phosphate pH 7.4, 100 μg/ml bovine γ globulin, 0.02% NaN3), applying 80 μl reaction volumes per black-bottomed 384 microtitre plate well (Nalge Nunc). 20 nM (corresponding to circa 20,000 fluorescence counts) of 5’FAM-labeled Strep-tagI (synthesized by GenScript Inc.), Strep-tag II or any novel hit were incubated with rising concentrations of Streptavidin/StrepTactin protein (in triplicates each) for at least 2 h at RT (or preferably o/n at 4 °C) to ensure binding equilibrium. Fluorescence emission (535 nm) intensities from both parallel and perpendicular orientations were after excitation at 480 nm measured in a DTX Multimode Detector (Beckman Coulter Genomics), checked for absence of abnormal intensity changes (that might result from solvent interference), and polarization values (expressed as millipolarization mP units) calculated via the formula mP = 1000(Is-GIp)/(Is + GIp), where G represents the G factor (= Is/Ip, settled as 0.650 for this study). Data were analyzed by Graphpad Prism 5 software, and the dissociation constant Kd calculated by non-linear regression (sigmoidal dose-response curve fitting).
Strep tag
Manufactured by GenScript
Sourced in United States
Strep-tag is a small peptide tag that can be fused to recombinant proteins to facilitate their purification and detection. The tag binds to Strep-Tactin, a modified streptavidin, allowing for the selective isolation of the tagged protein from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Py705 stat3, by Cell Signaling Technology (1 mentions)
Mln120b, by MedChemExpress (1 mentions)
Lamin b1, by Proteintech (1 mentions)
Amlexanox, by Selleck Chemicals (1 mentions)
Pyridone 6, by MedChemExpress (1 mentions)
Magstrep xt beads, by IBA Lifesciences (1 mentions)
Buffer bxt, by IBA Lifesciences (1 mentions)
Gapdh, by Proteintech (1 mentions)
α tubulin, by Proteintech (1 mentions)
Dh10bac e coli, by Thermo Fisher Scientific (1 mentions)
Superdex 200 increase 10 300 column, by GE Healthcare (1 mentions)
Cellfectin 2, by Thermo Fisher Scientific (1 mentions)
Microfluidization, by Microfluidics (1 mentions)
Sf9 cells, by Expression Systems (1 mentions)
Sf21 cells, by Expression Systems (1 mentions)
3 protocols using strep tag
1
Streptavidin-Streptag Binding Affinity Assay
2
Affinity Purification and Immunoblotting Assay
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Inhibitors MLN120B (MedChemExpress, Monmouth Junction, NJ, USA, HY-15473), Pyridone 6 (MedChemExpress, HY-14435), and Amlexanox (Selleck Chemicals, Houston, TX, USA, S3648); MagStrep XT beads (IBA Lifesciences, Göttingen, NI, Germany, 2-4090-002) and buffer BXT (IBA Lifesciences, 2-1041-250); recombinant human IL-6 protein (PeproTech, Rocky Hill, NJ, USA, 200-06-5); mouse monoclonal antibodies against GAPDH (Proteintech, Wuhan, Hubei, China, 60004-1-Ig) and V5 (Thermo Fisher Scientific, MA5-15253); rabbit monoclonal antibodies against pY705-STAT3 (Cell Signaling Technology, Danvers, MA, USA, 9145T); rabbit polyclonal antibodies against STAT3 (Proteintech, 10253-2-AP), α-Tubulin (Proteintech, 11224-1-AP), Lamin B1 (Proteintech, 12987-1-AP), and strep-tag (GenScript Biotech, Piscataway, NJ, USA, A00626-40) were purchased from the indicated companies. Rabbit polyclonal antibodies against LCMV NP were raised against NP protein (aa1-558).
3
Recombinant SARS-CoV-2 Spike Protein Production
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The SARS-CoV-2 sp12 gene was codon optimized and cloned into pFastBac with C-terminal additions of a TEV site and strep tag (Genscript). The pFastBac plasmid and DH10Bac E. coli (Life Technologies) were used to create recombinant bacmids. The bacmid was transfected into Sf9 cells (Expression Systems) with Cellfectin II (Life Technologies) to generate recombinant baculovirus. The baculovirus was amplified through two passages in Sf9 cells, and then used to infect 1 L of Sf21 cells (Expression Systems) and incubated for 48 hrs at 27°C. Cells were harvested by centrifugation, resuspended in wash buffer (25 mM HEPES pH 7.4, 300 mM NaCl, 1 mM MgCl2, 5 mM DTT) with 143 μL of BioLock per liter of culture. Cells were lysed via microfluidization (Microfluidics). Lysates were cleared by centrifugation and filtration. The protein was purified using Strep Tactin superflow agarose (IBA). Strep Tactin eluted protein was further purified by size exclusion chromatography using a Superdex 200 Increase 10/300 column (GE Life Sciences) in 25 mM HEPES, 300 mM NaCl, 100 μM MgCl2, 2 mM TCEP, at pH 7.4. Pure protein was concentrated by ultrafiltration prior to flash freezing in liquid nitrogen.
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