One hundred microliters of 10 μg/ml YadA in TBS was coated into a 96-well plate by incubation at RT for 1 h. The plate was washed three times with 200 μl TBS (20 mM Tris pH 7.5, 150 mM NaCl) and blocked using 200 μl of 3% BSA in TBS. Afterward, 100 μl of biotinylated heparin dilution (0–6.75 μg/ml) in TBS was added to the wells and incubated for 1 h at RT. The wells were washed three times with TBS as described above and blocked with 3% BSA in TBS for 1 h at RT. Strep-Tactin–HRP (IBA Lifesciences, 2-1502-001) at 1:1,000 was added in 3% BSA in TBS and incubated for 1 h at RT. The wells were washed again as described earlier, and an ABTS solution was used for detection as described before. After color development, the reaction was stopped by adding 100 μl of a 1% SDS solution. Absorbance at 405 nm was measured in a plate reader (BioTek Synergy H).
Strep tactin hrp
Manufactured by IBA Lifesciences
Sourced in Germany
Strep-Tactin–HRP is a fusion protein that combines Strep-Tactin, a variant of streptavidin, with horseradish peroxidase (HRP). It is designed for the detection and purification of Strep-tag fusion proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta, by Zeiss (2 mentions)
Synergy h, by Agilent Technologies (1 mentions)
Varioscan, by Thermo Fisher Scientific (1 mentions)
Goat anti human igg fc hrp, by Southern Biotech (1 mentions)
Biotin blocking buffer, by IBA Lifesciences (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
7 protocols using strep tactin hrp
1
YadA-Heparin Binding Assay Protocol
2
Integrin β1 Binding Assay
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96 wells (high-binding; 655061; Greiner Bio-One) were coated with 1 µM of His-Sumo–tagged or GST-tagged proteins in PBS (1.37 M NaCl, 26.8 mM KCl, 14.7 mM KH2PO4, and 78.1 mM Na2PO4) overnight at 4°C in triplicate. Wells were washed three times with PBS and blocked with 200 µl/well PBS plus 2% BSA for 1 h at RT before incubation with 0.5 µM of Strep-tagged integrin β1 proteins in PBS plus 0.05% Tween overnight at 4°C. For competitive assays with FLN19-21 and TalinF3 domain, Strep-tagged integrin β1 cytoplasmic domains were immobilized first before incubation with FLN19-21 and/or TalinF3. Wells were washed three times with PBS plus 0.05% Tween and blocked again for 1 h at RT. Streptactin-HRP (IBA Lifesciences) or mouse-α-His antibodies followed by HRP-conjugated goat α-mouse IgG antibodies for competitive assays were added in blocking buffer for 1 h at RT, respectively. Finally, wells were intensively washed with PBS plus 0.05% Tween, and 100 µl/well of tetramethylbenzidine (TMB) solution was added (9.5 ml of 2.4 mg/ml TMB in 1:9 aceton:ethanol plus 20 µl/10 ml H2O2 plus 0.5 ml 30mM potassium citrate, pH 4.1). The reaction was stopped after 20 min by adding 100 µl/well 2 M H2SO4, and samples were measured at 450 nm in a microplate reader (Varioscan; Thermo Fisher Scientific).
3
Siglec-Fc Quantification by ELISA
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Cells were split between 1:5 to 1:10 into T-175 flasks with 50–60 mL of media supplemented with 10 mM HEPES. Once cells reached confluency, the supernatant was harvested 7 days later by filtering the supernatant through a 0.22 μm filter. The filtered supernatant was stored at 4 °C until ready for use or purification. To estimate the amount of Siglec-Fc in the supernatant, an ELISA was carried out by coating wells overnight with anti-human IgG (Thermo Fisher) in phosphate-buffered saline (PBS). The following day, plates were washed in PBS-T (PBS containing 0.1% Tween-20) and incubated with PBS containing 5% BSA for 45 min. After five washes with PBS-T, the plate was then incubated for 1 hour with serial dilution of the supernatants. Serial dilutions were made in supernatants expressed from CHO Flp-in cells that had not been transfected, which showed no signal by ELISA. Following washing, wells were incubated for 1 h with Strep-Tactin-HRP (IBA Lifesciences) at a concentration of 0.1 μg/mL in PBS-T containing 1% BSA. After five washes of PBS-T, wells were developed with TMB Microwell Peroxidase Substrate System (KPL) for 15 min and quenched with 1 M phosphoric acid and read at 450 nm. Purified protein (see below) was used to generate a standard curve to estimate the amount of protein in the supernatants.
4
Evaluating Stress-Induced SARS-CoV-2 Spike Decay
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ELISAs were in either a spike or antibody capture configuration as indicated. For spike capture, plates were coated with 1 μg/mL of purified spike proteins in PBS. Duplicate serial dilutions of each full-length antibody in PBS-T with 5% milk were allowed to bind each coat, and the secondary antibody solution was a 1:1200 dilution of goat-anti-human IgG Fc-HRP (SouthernBiotech). For antibody capture, antibody was coated at 1 μg/mL in PBS. Duplicate serial dilutions of spike in PBS-T with 3% bovine serum albumin were allowed to bind each coat, and the secondary antibody solution was a 1:2000 dilution of streptactin-HRP (IBA Lifesciences). ELISA curves were fit to a four-parameter logistic curve.
Fresh aliquots of SARS-2 and SARS-2 HexaPro spikes were thawed and split to stress the spike proteins. One half of the aliquot was stressed by incubation at –20°C for 5 min, then 50°C for 2 min for three temperature cycles. The freshly thawed and stressed spikes were evaluated in antibody capture ELISAs. For each fresh and stressed spike, 8 µg was analyzed by SDS-PAGE under non-reducing conditions.
Fresh aliquots of SARS-2 and SARS-2 HexaPro spikes were thawed and split to stress the spike proteins. One half of the aliquot was stressed by incubation at –20°C for 5 min, then 50°C for 2 min for three temperature cycles. The freshly thawed and stressed spikes were evaluated in antibody capture ELISAs. For each fresh and stressed spike, 8 µg was analyzed by SDS-PAGE under non-reducing conditions.
5
Confocal Microscopy and Immunoblotting Protocol
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Confocal microscopy was performed using a Zeiss LSM 510 META laser scanning microscope (Carl Zeiss, Thornwood, NY, USA). Argon laser line excitation wavelength and emission bandpass filter wavelengths for mTq2 and mVenus were 458 nm and 480–520 nm, and 488 nm and 500–550 nm, respectively. Chlorophyll autofluorescence was detected, in parallel with mTq2 acquisition, using a 650–710 nm bandpass filter. Image acquisition parameters (e.g. laser power, pinhole, detector gain, etc.) and sampling time post-infiltration were held constant within an experiment (i.e. within each figure). Raw BiFC data for the mVenus channel were not altered beyond equal signal increases (by equally decreasing the lower dynamic range value) in the zeiss zen 2010 software (www.zeiss.com ) within an experiment, and subtraction of autofluorescence where noted.
Immunoblots were performed using a ThermoFisher FLAG–HRP antibody (FG4R, MA1–91878,www.thermoscientific.com ) and IBA StrepTactin–HRP (2-1502-001, www.iba-lifesciences.com ) and the manufacturer's Strep-tag detection protocol. Split-ubiquitin assays were performed as previously detailed (Gookin and Bendtsen, 2013 (link)).
Immunoblots were performed using a ThermoFisher FLAG–HRP antibody (FG4R, MA1–91878,
6
Western Blot Analysis of Cell Lysates
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Lysates of KC or C6/36 cells were analysed by SDS-PAGE (10% polyacrylamide gel) and then electro-blotted onto a 0.45 μm nitrocellulose membrane as previously described [23 (link),26 (link)]. Membranes were blocked with 20 mL of blocking buffer (PBS 1X, 3% BSA and 0.05% Tween20) at room temperature for 1 h. This was followed by three washes with PBS containing 0.1% Tween20 (PBST). In some experiments, membranes were further blocked by diluting 10 µL of biotin blocking buffer (IBA Lifesciences, Göttingen, Germany) in 10 mL of PBS containing 0.1% Tween20.
Strep-Tactin® HRP (IBA Lifesciences, Göttingen, Germany) was diluted 1/100 in a buffer containing 100 mM Tris-HCl pH 8.8, 100 mM NaCl and 5 mM MgCl2. Ten microliters of this preparation were further diluted in 10 mL PBST prior to being added to the membrane, which was incubated for 1 h at room temperature. Membranes were washed twice with PBST for 1 min and then twice with PBS for 1 min at room temperature. Proteins on membranes were revealed by chemiluminescence detection using Clarity™ Western ECL Substrate (Bio-Rad, Marnes-la-Coquette, France). Blots were imaged using the Bio-Rad Chemidoc XRS+.
Strep-Tactin® HRP (IBA Lifesciences, Göttingen, Germany) was diluted 1/100 in a buffer containing 100 mM Tris-HCl pH 8.8, 100 mM NaCl and 5 mM MgCl2. Ten microliters of this preparation were further diluted in 10 mL PBST prior to being added to the membrane, which was incubated for 1 h at room temperature. Membranes were washed twice with PBST for 1 min and then twice with PBS for 1 min at room temperature. Proteins on membranes were revealed by chemiluminescence detection using Clarity™ Western ECL Substrate (Bio-Rad, Marnes-la-Coquette, France). Blots were imaged using the Bio-Rad Chemidoc XRS+.
7
SARS-CoV-2 Antibody Detection Assay
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Cell supernatant containing secreted antibodies was analyzed for the presence of coronavirus specific responses. First, supernatant was collected and filtered with 0.2 μm filters (Sartorius, 16534-K). 96-well microtiter plates were coated with recombinant coronavirus antigens purchased from SinoBiological: SARS2-Spike S1+S2 (40,589-V08B1), SARS1-S1 (40,150-V08B1), MERS-CoV Spike (40,069-V08B), HCoV-229E Spike (40,605-V08B), HCoV-OC43 Spike (40,607-V08B), HCoV-HKU1 Spike (40,606-V08B), HCoV-NL63 Spike (40,604-V08B), SARS-CoV-2 Nucleocapsid (40,588-V08B) at 8 μg/mL in PBS at 4°C overnight. After blocking with 2% milk, 0.05% tween PBS (PBSTM) for 1 h at RT, cell supernatants were diluted 1:2 in 2% milk PBS (PBSM) and added to the microtiter plate for 1 h at RT. Antigen-specific responses were detected using Strep-Tactin®-HRP (iba lifesciences, #2-1502-001) diluted 1:10′000 in PBSM, followed by repeated washing and using TMB (3,3′,5,5′-tetramethylbenzidine) (1-Step™ Ultra TMB, #34028) as a substrate. The absorbance of each sample was measured at 450 nm as well as 630 nm. All samples reported here were interrogated in the same plate.
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