Proteins were separated by NuPAGE 4–12% Bis-Tris gels (ThermoFisher Scientific) and transferred to nitrocellulose membranes (Amersham Protran Premium, 0.45 μm). The membranes were blocked with 5% nonfat dry milk (Sema5ATSR3-4 proteins with biotinylated Avitag) or 3% Bovine Serum Albumin (His-tagged Sema5Asema-TSR1-7 proteins) in PBS for 1 h at room temperature. For His6 tag detection, the membranes were incubated with primary antibody (6xHis Monoclonal Antibody, TaKaRa, cat. no. 631212 dilution 1:3000) for 1 h at room temperature. Blots were then washed six times for 5 min with PBS-0.1% Tween-20 and incubated for 1 h at room temperature with secondary antibody conjugated to horseradish peroxidase (Anti-mouse IgG peroxidase polyclonal goat antibody, Sigma, cat. no. A0168, dilution 1:10,000). For biotinylated Avitag detection, the blot was incubated with Streptactin HRP (BioRad) antibody at 1:25,000 dilution for 1 h at room temperature. This was followed by washing of blots six times for 5 min with PBS-0.1% Tween-20, and signal detection using ECL (BioRad).
Streptactin hrp
Manufactured by Bio-Rad
Sourced in United States
Streptactin HRP is a streptavidin-horseradish peroxidase conjugate used in various biotechnological applications. It provides a high-affinity interaction with biotin-labeled molecules, enabling the detection and quantification of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Protran, by Cytiva (2 mentions)
6xhis monoclonal antibody, by Takara Bio (1 mentions)
Anti mouse igg peroxidase polyclonal goat antibody, by Merck Group (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (1 mentions)
Precision plus protein westernc, by Bio-Rad (1 mentions)
Immobilon western, by Merck Group (1 mentions)
Bira biotin protein ligase standard reaction kit, by Avidity (1 mentions)
Sa3800 spectral cell analyzer, by Sony (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Ponceau s, by Merck Group (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Amersham ecl western blotting detection kit, by GE Healthcare (1 mentions)
Amersham ecl western blotting, by Cytiva (1 mentions)
7 protocols using streptactin hrp
1
Western Blot Analysis of Sema5A Proteins
2
Immobilized Streptavidin Binding Assay
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Streptavidin type II (1 μg/mL in 100 μL PBS, Fujifilm-Wako, Osaka, Japan) was immobilized in transparent microplate wells (Greiner 655061, Tokyo, Japan) at 4°C overnight. The wells were blocked with 20% Immunoblock (DS Pharma, Osaka, Japan) and washed three times with PBST (PBS containing 0.1% Tween 20). HLA-DRB1∗11:01, HLA-DRB1∗15:01, or HLA-DRB1∗09:01 complexed with biotinylated HLA-DRA∗01:01 and CLIP (4 μg/mL; ProImmune, Oxford, UK) in 100 μL PBS was added and incubated for 1 h at 25°C. The wells were then washed three times with PBST and incubated with purified C-terminally StreptagII-fused MVR-scFv (1 μg/mL in 100 μL PBS) or PBS for 1 h at 25°C. After washing with PBST, 5,000-fold diluted Streptactin:HRP (BioRad, Hercules, CA, USA) was applied and incubated for 30 min at 25°C. The wells were washed with PBST and measured for HRP activity using a substrate (100 μg/mL 3,3′5,5′-tetramethylbenzidine and 0.02% H2O2 in 100 mM sodium acetate, pH 6.0).
3
Immunoblotting Analysis of Cardiac Biopsy Samples
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Sections of unfixed cardiac biopsies, both untreated and decellularised, were analysed by immunoblotting. Blotted-dry tissue was ultrasonicated in the presence of Laemmli buffer (~ 10 μg tissue/μl). After heating at 95 °C for 5 min and centrifugation for 10 min at 10,600g, reduced denatured supernatants (3 μl) were subjected to SDS-PAGE in precast 15% gradient polyacrylamide gels (Excel, GE Healthcare, Little Chalfont, UK) and then blotted onto an activated PVDF membrane. Polyclonal rabbit anti-κ light chain (4 μg/ml, Dako, Glostrup, Denmark) or sheep anti-human TTR (3 μg/ml, The Binding Site, Birmingham, UK) antibodies were used to identify their cognate antigens and were detected respectively by peroxidase-conjugated polyclonal sheep anti-rabbit (25 × 10− 3 μg/ml, Dako, Glostrup, Denmark) or anti-sheep (13 × 10− 3 μg/ml) immunoglobulins, followed by 1:10,000 StrepTactin-HRP (Biorad, Hercules, CA, USA) for identification of the molecular weight standards (Precision Plus Protein WesternC, Biorad, Hercules, CA, USA). Peroxidase activity was visualized using a chemiluminescent substrate (Immobilon Western, Millipore, Billerica, MA, USA) and a FluorChem M system (Proteinsimple, San Jose, CA, USA).
4
Biotinylation and Flow Cytometry of Sema5A
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Sema5ATSR3-4 constructs containing a C-terminally fused 3C-Avi-His6 tag were produced in HEK 293T cells, purified from the secreted medium by Ni-NTA, and site-specifically biotinylated at the AviTag conjugated to their C-terminus in vitro, using a BirA biotin-protein ligase standard reaction kit Avidity LLC (CO, USA, avidity.com). Successful biotinylation was confirmed by Western blot incubated with Streptactin HRP (BioRad) antibody at 1:25,000 dilution (Supplementary Fig. 1j ). For each assay sample, 1 × 105 of genetically engineered CHO GS-/- cells33 (link),34 (link) were harvested and washed in PBS before being resuspended in 50 µg/mL of biotinylated WT, R747E/R749E or K734E/R747E/R749E Sema5ATSR3-4 diluted in PBS with added 1% FBS (assay buffer), gently shaking for 1 h at 4 °C. The cells were then washed with assay buffer before incubation with Alexa Flour 488-streptavidin (1:2000, #S32354, Invitrogen) diluted in assay buffer while gently shaking for 30 min at 4 °C. After wash in assay buffer, the cells were resuspended in assay buffer and subjected to flow cytometry on a SA3800 spectral cell analyzer (SONY), where mean fluorescent intensity for each sample was measured. All experiments were performed a minimum of 3 times using triplicate samples, and mean fluorescent intensity was normalized to CHO WT for all samples.
5
Western Blot Analysis of α-Synuclein
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Cells were seeded and grown to 60% confluency and differentiated in T25 culture flask (Greiner, Germany). Cells were then lysed [4× Laemmli buffer (8% SDS, 240 mM Tris-Cl, pH 6.8), 100 mM DTT] for 15 minutes at room temperature, scraped and transferred to Eppendorf tubes. Lysates were sonicated, boiled for 5 minutes and centrifuged for 2 minutes at 12000 rcf (IEC MicroMAX tabletop centrifuge). Cleared lysates were separated on a SDS-PAGE gel (12%) and blotted onto methanol activated PVDF membrane (Millipore, USA). For improved α-S detection, membranes were first fixed in a 0.4% PFA in PBS solution, then blocked in non-fat-dried-milk (5% ELK, Campina) in TBS and Tween-20 (0.3%) and incubated overnight at 4 °C with primary antibody against α-S or GAPDH. Primary antibodies were detected by goat-anti-mouse-HRP conjugated antibodies [1:8000 in TBS + tween-20 (0.3%)]. Protein Marker bands were visualized by Strep-Tactin-HRP [1:5000 in TBS + tween-20 (0.3%)] (Bio-Rad, USA). HRP-conjugated protein bands were detected by Clarity Western ECL Substrate (BioRAD, USA) and imaged on a FluorChem M imager (Protein Simple, USA).
6
Western Blot Analysis of Adherent Cells
7
Quantitative sgRNA expression analysis
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A serial dilution of sgRNA probes and positive control were dropped onto the nylon membrane, which was UVcross-linked twice using the Auto-crosslink mode in a Stratalinker 2400 UV Crosslinker. The membrane was then blocked in blocking buffer (1× phosphate-buffered saline [PBS] pH 7.4, 1% [w/v] sodium dodecyl sulfate [SDS]) for 30 min, and incubated with StrepTactin-HRP (1:5,000; Bio-Rad, Hercules, CA, USA; Cat: #1610381) diluted in blocking buffer for 30 min. After washing the membrane once in 0.1× blocking buffer and twice in 1× PBST buffer (1× PBS pH 7.4 with 0.05% [v/v] Tween-20), ECL substrate was added for chemiluminescent detection.
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