Flat-bottom 96-well ELISA plates were incubated overnight with 0.25 mg/ml fulllength CSP protein or 1.0 mg/ml Streptavidin (Roche) in a humidity chamber at 22 C. Streptavidin-coated plates were then incubated with 2.0 mg/ml biotinylated NANP6 or Pf16 peptide. All wells were subsequently blocked with 0.5% casein for 1 hour at 22 C, then incubated with serially diluted serum samples at 22 C for 2 hours. Plates were then incubated with PBS or 4M Urea for 1 hour at 22 C. Wells were then developed by incubating with peroxidase-labeled goat anti-human IgG (KPL) followed by ABTS peroxidase substrate (KPL). Data were collected using Soft-Max GxP and fit to a 4-parameter logistic curve. For each sample, the serum dilution corresponding to an optical density (OD) of 1.0 was calculated with and without the presence of urea. The avidity index was then calculated by dividing the serum titer (at OD = 1.0) obtained in the presence of the urea by the serum titer obtained in the presence of PBS.
Streptavidin
Manufactured by Roche
Sourced in Germany, France, United States
Streptavidin is a protein derived from the bacterium Streptomyces avidinii. It has a high affinity for the vitamin biotin, forming a strong, non-covalent bond. Streptavidin is commonly used in various biotechnology and laboratory applications where the biotin-streptavidin interaction is utilized.
Automatically generated - may contain errors
Lab products found in correlation
Microarray chip, by Thermo Fisher Scientific (1 mentions)
Microarray suite 5, by Thermo Fisher Scientific (1 mentions)
Streptavidin phycoerythrin, by Thermo Fisher Scientific (1 mentions)
Goat igg, by Merck Group (1 mentions)
Eukaryotic hybridization controls, by Thermo Fisher Scientific (1 mentions)
Nbt bcip, by Roche (1 mentions)
Centricon, by Merck Group (1 mentions)
Nhs biotin, by Merck Group (1 mentions)
Biacore t200, by Cytiva (1 mentions)
Protease, by Merck Group (1 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
1 dik, by Mabtech (1 mentions)
Bcip nbt substrate, by Merck Group (1 mentions)
Alkaline phosphatase, by Roche (1 mentions)
Interleukin 2 (il 2), by GE Healthcare (1 mentions)
L glutamate, by Thermo Fisher Scientific (1 mentions)
Scd40l, by Enzo Life Sciences (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Il 10, by R&D Systems (1 mentions)
9 protocols using streptavidin
1
Avidity Assay for Anti-Malaria Antibodies
2
Comparative Microarray Hybridization Protocols
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Example 4
NZ tumours: Hybridisation of the labelled target cDNA was performed using MWG Human 30K Array oligonucleotides printed on epoxy coated slides. Slides were blocked with 1% BSA and the hybridisation was done in pre-hybridisation buffer at 42° C. for at least 12 hours followed by a high stringency wash. Slides were scanned with a GenePix Microarray Scanner and data was analyzed using GenePix Pro 4.1 Microarray Acquisition and Analysis Software (Axon, CA).
DE tumours: cRNA was mixed with B2-control oligonucleotide (Affymetrix, Santa Clara, Calif.), eukaryotic hybridization controls (Affymetrix, Santa Clara, Calif.), herring sperm (Promega, Madison, Wis.), buffer and BSA to a final volume of 300 μl and hybridized to one microarray chip (Affymetrix, Santa Clara, Calif.) for 16 hours at 45° C. Washing steps and incubation with streptavidin (Roche, Mannheim, Germany), biotinylated goat-anti streptavidin antibody (Serva, Heidelberg, Germany), goat-IgG (Sigma, Taufkirchen, Germany), and streptavidin-phycoerythrin (Molecular Probes, Leiden, Netherlands) was performed in an Affymetrix Fluidics Station according to the manufacturer's protocol. The arrays were then scanned with a HP-argon-ion laser confocal microscope and the digitized image data were processed using the Affymetrix® Microarray Suite 5.0 Software.
3
Recombinant Protein Binding Assay
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Plasma proteins binding was assayed by western blotting, using biotinylated human serum proteins, including fibrinogen, fibronectin, lactoferrin and mucin (12 (link)). The recombinant protein was electroblotted on nitrocellulose membrane as previously mentioned. After blocking and washing the membrane, it was incubated in plasma proteins diluted in 1:1000 for 3 hours at room temperature. Then, the membrane was washed as before, incubated in Streptavidin (Roche, Germany), and diluted in washing buffer in 1:30000 at room temperature for 3 hours. Subsequently, the membrane was washed and incubated in alkaline phosphatase buffer for 5 minutes. Then, the membrane was immersed in NBT/BCIP (Roche, Germany) solution and kept in dark condition until the protein band appeared. The ScaB recombinant protein was used as negative control.
4
Efficient Biotinylation of Bacterial Effectors
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Fractions of the antigens EHEC TirM or EPEC TirM were diluted in 1.5 mL PBS to 1 mg ml−1. The NHS-Biotin (Sigma-Aldrich B2643) was dissolved in DMSO and immediately added to these protein solutions in a biotin:protein molar ratio of 20:1 and the mix was incubated for 30 min at RT with gentle agitation. Then 1 M Tris-HCl, pH7.5 was added for a final concentration of 50 mM and the mixture was incubated for 1 h on ice. The sample was concentrated to a final volume of 500 µL in a 3 kDa Centricon (Merck) by centrifugation at 5000 g. Finally, the efficiency of biotinylation was checked in a Western blot revealed with POD-anti-biotin (horseradish peroxidase conjugated with Streptavidin, Roche).
5
Biacore-Based Streptavidin-Biotin RNA Binding
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SPR experiments were performed at 10 °C using a Biacore™ T200 (Biacore™, GE Healthcare Lifesciences, Upsala, Sweden). A HC200 m sensor chip (XantecBioanalytics, Düsseldorf, Germany) was coated with 5000 resonance units (RU) of streptavidin (Roche Applied Sciences, Meylan, France) using the Biacore™ amine-coupling kit. 5′ biotinylated RNAs (150 RU) were immobilized on one flow cell. One blank flow cell was used for double-referencing of the sensorgrams using BiaEval 4.1 (Biacore™). The RNA samples injected over the functionalized surface, prepared in running buffer (10 mM sodium phosphate buffer, pH 7.2 at 20 °C, 50 mM NaCl, 3 mM MgCl2, 0.05 % Tween-20), were heated for 1 min and 30 s at 90 °C, and then put on ice for 5 min. The samples were injected at 50 µL/min. The regeneration of the surface was achieved using a 2-min pulse of a mixture of 40 % formamide, 3.6 M urea and 30 mM EDTA, prepared in Milli-Q water [26 ].
6
T Cell Receptor Crosslinking Activation
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For each condition, 2 × 106in vitro activated T cells were incubated on ice with 20 μg/mL biotinylated anti-CD3 antibodies (145-2C11, eBioscience) in serum-free RPMI medium for 30 min, washed twice, and pre-warmed at 37 °C for 5 min. To crosslink TCRs, 10 μg of pre-warmed streptavidin (Roche) were added to T cells for the indicated length of time. The cells were subsequently lysed by the addition of equal volume of ice-cold lysis buffer (100 mM Tris (pH 8.0), 2% Triton X-100, 4 mM EDTA) supplemented with protease (Sigma) and phosphatase inhibitors (Thermo Scientific).
7
ELISPOT Assay for IFN-γ Secretion
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ELISPOT plates (MSIPN4550, Millipore) were prewet and washed with PBS, and coated overnight at 4°C with anti‐IFNγ antibody (1‐DIK, Mabtech). Plates were washed using PBS and then saturated with RPMI complemented with 10% FBS. Plates were washed and HeLa‐CIITA cells (105 cells/well) were co‐cultured with T cell clones (5.103 and 1.103 cells/well) overnight at 37°C. Cells were removed and plates were then washed with PBS‐0,05% Tween‐20 prior to incubation with biotinylated anti‐IFNγ antibody (7‐B6‐1, Mabtech) (2 h, RT). Spots were revealed using alkaline‐phosphatase coupled to streptavidin (0,5 U/ml, Roche Diagnostics) (1 h, RT) and BCIP/NBT substrate (B1911, Sigma‐Aldrich) (30 min, RT). Reactions were stopped using water. A number of spots were counted using AID reader (Autoimmun Diagnostika GmbH). For each experimental condition, ELISPOTs were performed mostly in triplicates or at least in duplicates.
8
CLL Cell Stimulation and Cytospin Preparation
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Two million viable primary CLL cells per milliliter were grown in RPMI medium supplemented with 10% FBS (Gibco), 1% PEST (Gibco), and 1% l -glutamate (Gibco), supplemented with 25% vol/vol serum-free supernatant from the T cell hybridoma cell line MP6 (Rosén et al., 1986 (link)), as a source of thioredoxin (Söderberg et al., 1999 (link); Nilsson et al., 2000 (link)). For stimulation of the BcR, the modified RPMI medium was supplemented with 3 µg/ml AffiniPure F(ab′)2 fragment rabbit anti–human IgM (Jackson ImmunoResearch Laboratories, Inc.), 10 ng/ml IL-2 (GE Healthcare), and 1 µg/ml streptavidin (Roche). To stimulate the CD40 signaling pathway, cells were treated with the modified RPMI medium supplemented with 100 ng/ml sCD40L (Enzo Life Sciences), 25 ng/ml IL-4 (R&D Systems), and 100 ng/ml IL-10 (R&D Systems). Cells were stimulated for 15 min at 37°C (5% CO2). Cytospins were prepared using 150,000 cells per slide and a Cellspin I cytocentrifuge (Tharmac) at 500 rpm for 2 min.
9
Quantification of Amyloid-beta 1-42 by ELISA
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The levels of Aβ 1‐42 were performed by ELISA kit as previously described 38 . In brief, microtiter plates (Maxisorp; Nunc, Termo Fisher Scientific, Waltham, MA, USA) were sensitized with streptavidin (Roche Biochemicals, Roche Diagnostics S.p.A. Monza (MB), Italy) overnight. Primary capture antibody, biotinylated 6E10 (1 mg/ml; Senetek, Maryland Heights, MO, USA) was added for 8 hrs. Concentrated (50‐fold) cell culture supernatants were diluted to 1 mg/ml with assay buffer [50 mM Tris‐HCl (pH 7.5)] containing 140 mM NaCl, 5 mM EDTA, 0.05% Nonidet P‐40, 0.25% gelatin and 1% bovine serum albumin and incubated for 24 hrs at 4°C. The BAP‐15 antibody specific for Aβ1–42 was used. After colour development with tetramethylbenzidine (Roche Biochemicals), the plate was analysed on a Lab systems Multiskan RC plate reader using Genesis software (Lab systems). The test was performed in triplicate.
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