Anti-EGFR, GAPDH and 14-3-3ζ were from Santa Cruz Biotechnology. Anti-Pan-AKT, anti-AKT pSer473, anti phosphoEGFR antibodies were from Cell Signaling Technology. HA-peroxidase and anti-PTP1B (FG6) was from Millipore. PT-66-agarose-conjugated beads, anti-FLAG M2 beads, and anti-HA beads and anti-Flag M2 peroxidase were purchased from Sigma. Anti-PTP1B pSer50 (Ab62320) were from Abcam. Streptavidin-HRP was from GE Healthcare. HRP-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. Protease inhibitor mixture tablets were from Roche. Catalase and superoxide dismutase were from Calbiochem. Surfact-Amps Nonidet P-40, zeba desalt spin columns, EZ-Link biotin-iodoacetyl-PEG2 (biotin-IAP), and iodoacetic acid were from ThermoScientific. The pTyr loop-derived peptide (CKNRNRYRDVS) and phospho-Ser50 pTyr loop-derived peptide (CKNRNRYRDVpS) were from GenScript USA Inc. BIACore sensor NTA and Streptavidin chips were from GE healthcare.
Streptavidin chip
Manufactured by GE Healthcare
Sourced in United States
The Streptavidin chip is a laboratory equipment used for the immobilization and detection of biotinylated molecules. It utilizes the high-affinity interaction between streptavidin and biotin to capture and immobilize target analytes on a solid surface. The chip provides a platform for various analytical and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti ptp1b fg6, by Merck Group (2 mentions)
Pt 66 agarose conjugated beads, by Merck Group (2 mentions)
Anti ptp1b pser50 ab62320, by Abcam (2 mentions)
Surfact amps nonidet p 40, by Thermo Fisher Scientific (2 mentions)
Ez link biotin iodoacetyl peg2 biotin iap, by Thermo Fisher Scientific (2 mentions)
Biacore sensor nta, by GE Healthcare (2 mentions)
Anti flag m2 peroxidase, by Merck Group (2 mentions)
Anti pan akt, by Cell Signaling Technology (2 mentions)
Anti pser473 akt, by Cell Signaling Technology (2 mentions)
Streptavidin hrp, by GE Healthcare (2 mentions)
Zeba desalt spin column, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor mixture tablet, by Roche (2 mentions)
Anti flag m2 bead, by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Ha peroxidase, by Merck Group (2 mentions)
Iodoacetic acid, by Thermo Fisher Scientific (2 mentions)
Anti egfr, by Santa Cruz Biotechnology (2 mentions)
Anti phospho egfr, by Cell Signaling Technology (2 mentions)
Anti ha beads, by Merck Group (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
8 protocols using streptavidin chip
1
Protein Interaction and Modification Analysis
2
Protein Interaction and Modification Analysis
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Anti-EGFR, GAPDH and 14-3-3ζ were from Santa Cruz Biotechnology. Anti-Pan-AKT, anti-AKT pSer473, anti phosphoEGFR antibodies were from Cell Signaling Technology. HA-peroxidase and anti-PTP1B (FG6) was from Millipore. PT-66-agarose-conjugated beads, anti-FLAG M2 beads, and anti-HA beads and anti-Flag M2 peroxidase were purchased from Sigma. Anti-PTP1B pSer50 (Ab62320) were from Abcam. Streptavidin-HRP was from GE Healthcare. HRP-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. Protease inhibitor mixture tablets were from Roche. Catalase and superoxide dismutase were from Calbiochem. Surfact-Amps Nonidet P-40, zeba desalt spin columns, EZ-Link biotin-iodoacetyl-PEG2 (biotin-IAP), and iodoacetic acid were from ThermoScientific. The pTyr loop-derived peptide (CKNRNRYRDVS) and phospho-Ser50 pTyr loop-derived peptide (CKNRNRYRDVpS) were from GenScript USA Inc. BIACore sensor NTA and Streptavidin chips were from GE healthcare.
3
Affilin Binding Kinetics Evaluation
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Example 5
After serum incubation, surface plasmon resonance (SPR) measurements on a Biacore 3000 (GE Healthcare) were used to determine the binding behavior of Affilin-138800 and Affilin-77404 to target 67689 and off-target 6789. The protein was captured from serum incubation after 1 h and 24 using streptactin matrix (IBA, Goettingen). Biotinylated target and off target were immobilized on a streptavidin chip (GE Healthcare) followed by concentration dependent injection of Affilin molecules (0-500 nM) in PBST. All 67689-traces were corrected by subtraction of the 6789 flow channel. KD, kon and Koff values were calculated by fitting the traces using a global kinetic fitting (1:1 Langmuir model, BIAevaluation 3.0 software). Results are shown in
4
Heparin-proMMP7 Interaction Kinetics
5
TCR-pMHC Binding Affinity Measurement
6
Quantification of Soluble Selectin Levels
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LE-His was quantified by a sandwich ELISA using DREG-200 as capture and biotinylated DREG-55 (self-prepared from hybridoma with subsequent biotinylation) as detection antibody following standard procedures, whereas human serum (Thermo Fisher Scientific, Dreieich, Germany) served as a standard.
Selectin concentration from the cell culture supernatant was additionally determined using calibration free concentration analysis provided by the Biacore X100 with plus package (GE Healthcare, Freiburg, Germany). Here, only the active protein that is able to bind the aptamer is considered. In this set-up, samples are injected at flow rates of 10 µL/min and 100 µL/min under mass transfer limitation. Therefore, biotinylated LD201mΔ1 was coupled to 1900 RU to a streptavidin chip (GE Healthcare, Freiburg, Germany). The reference lane was left untreated. By providing information about the mass (32,114 Da) and the diffusion coefficient (8.5 × 10−11 m2·s−1) of the analyte, the built-in software of the Biacore X100 calculates protein concentrations from the slope differences [15 (link),16 (link),17 (link)]. HBS-Ca (20 mM HEPES, pH 7.5, 150 mM NaCl and 1 mM CaCl2) was used as running buffer, 100 mM EDTA as regeneration buffer and cell culture medium of untransfected HEK cells was used as a blank.
Selectin concentration from the cell culture supernatant was additionally determined using calibration free concentration analysis provided by the Biacore X100 with plus package (GE Healthcare, Freiburg, Germany). Here, only the active protein that is able to bind the aptamer is considered. In this set-up, samples are injected at flow rates of 10 µL/min and 100 µL/min under mass transfer limitation. Therefore, biotinylated LD201mΔ1 was coupled to 1900 RU to a streptavidin chip (GE Healthcare, Freiburg, Germany). The reference lane was left untreated. By providing information about the mass (32,114 Da) and the diffusion coefficient (8.5 × 10−11 m2·s−1) of the analyte, the built-in software of the Biacore X100 calculates protein concentrations from the slope differences [15 (link),16 (link),17 (link)]. HBS-Ca (20 mM HEPES, pH 7.5, 150 mM NaCl and 1 mM CaCl2) was used as running buffer, 100 mM EDTA as regeneration buffer and cell culture medium of untransfected HEK cells was used as a blank.
7
Kinetic Analysis of USP1 Binding to DNA
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SPR binding experiments were carried out in the Biacore T200 system (GE, USA) to test the binding of USP1 WT and mutants with double‐stranded DNA. The running buffer used for the SPR experiment was 20 mM HEPES pH 7.5, 150 mM NaCl, 2 mM DTT, 0.05% Tween‐20, 1 mg ml−1 BSA and the DNA was immobilized on a Streptavidin chip (Sensor chip SA, GE) using the biotin present on the 5′ end of the DNA. The binding experiments were carried out in the single cycle kinetics mode with 10 sequential injections of USP1 and USP1ΔL1 from 25 to 0.05 µM, while the insert 1 and mutants were injected from 100 to 0.2 µM. Data from a reference flow cell (−DNA) which was run in parallel to the experiment were subtracted from the signal using the Biacore T200 evaluation software. The final analysis and figures were done in GraphPad Prism 7 software (GraphPad Software Inc, USA).
8
Anti-CD28 dAb Affinity Characterization
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The anti-CD28 dAb (BMS-1m74-14982) precursor was identified by phage display from a library of human heavy and light chain dAbs. The identified Vκ chain then underwent random mutagenesis followed by site-directed mutagenesis in the complementarity-determining regions and was selected for increased affinity for murine CD28 using a surface plasmon resonance assay. To determine affinity and kinetics, a streptavidin chip (GE Healthcare) surface was preconditioned with 1 M NaCl and 50 mM NaOH. Mouse and human CD28-biotinylated monomers (1 µg/ml) were immobilized on a flow cell with 15-s contact time at 10 µl/min to give ∼180 RUs. The mouse anti-CD28 dAb was injected at different concentrations (800–1.56 nM) for 3 min at 30 µl/min followed by 15 min of dissociation. Surfaces were regenerated with two 45-s pulses of 10 mM Glycine. Kinetic analysis was performed using the Biacore T100 evaluation software using a global analysis 1:1 Langmuir binding model. Calculation of the average kinetic parameters and standard errors was determined using three replicates (Table 1 ). The anti-CD28 dAb is formatted with a 40-kD branched polyethylene glycol, and had an EC50 (2.2 ± 0.6 nM) comparable to that of human CTLA4Ig (abatacept, 4.25 ± 2 nM) in murine mixed lymphocyte reactions.
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