The samples were diluted in SDS-containing buffer and run on 4–20% Tris-Glycine polyacrylamide gels (Invitrogen). To reduce the disulfide bonds, some samples were treated with β-mercaptoethanol for 3 min at 95°C before electrophoresis. SeeBlue Plus2 Pre-Stained Standard (Invitrogen) was used to indicate the molecular weights. The proteins were blotted onto a polyvinyl fluoride membrane (Biorad) by 100 V for 1 h at 4°C. Milk and casein (5% and 1%, respectively) in 0.1% Tween 20 was used to block the membrane before incubation over night at 4°C with biotinylated anti-mCherry followed by incubation with streptavidin-conjugated HRP (Amersham Bioscience). The protein bands were developed by a chemiluminescent peroxidase substrate, Lumigen (GE Healthcare) and images were acquired by Kodak image station 2000R.
Hrp conjugated streptavidin
Manufactured by Cytiva
Sourced in United Kingdom
HRP-conjugated streptavidin is a protein complex composed of streptavidin covalently linked to horseradish peroxidase (HRP). It is commonly used as a detection reagent in various bioassays and immunoassays that rely on the high-affinity interaction between streptavidin and biotin.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinyl fluoride membrane, by Bio-Rad (1 mentions)
Seeblue plus2 pre stained standard, by Thermo Fisher Scientific (1 mentions)
Tris glycine polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Lumigen, by GE Healthcare (1 mentions)
Image station 2000r, by Kodak (1 mentions)
Synergy ht microplate reader, by Agilent Technologies (1 mentions)
Cystamine, by Merck Group (1 mentions)
Maxisorp elisa plate, by Thermo Fisher Scientific (1 mentions)
Elx808 absorbance microplate reader, by Agilent Technologies (1 mentions)
Tetramethylbenzidine tmb substrate, by BD (1 mentions)
5 protocols using hrp conjugated streptavidin
1
Western Blot Analysis of mCherry
2
Quantification of Active β1 Integrin
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Corning 96-well clear polystyrene high bind stripwell microplates (Cat #2592) were coated overnight with 5 µg/ml of rat mAb anti-active β1 integrin clone 9EG7 in 0.05 M Na2CO3 (pH 9.6) at 4°C and were next blocked in PBS containing 0.05% Tween-20 (PBS-T) and 5% BSA for 1 h at RT. Internalized sulfo-NHS-SS–biotinylated 9EG7+ active β1 integrins were captured by overnight incubation of 50 µl EC lysates at 4°C. Unbound material was removed by extensive washing with PBS-T. Wells were then incubated with streptavidin-conjugated HRP (Amersham) in PBS-T containing 1% BSA for 1 h at 4°C. After further washing, internalized sulfo-NHS-SS–biotinylated 9EG7+ active β1 integrins were detected by a chromogenic reaction with ortho-phenylenediamine that was quantified by means of a Synergy HT microplate reader (at 490 nm; BioTek Instruments) and the Gen5 software (BioTek Instruments).
3
Quantifying Transglutaminase Activity in Tissues
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A dot blot assay was used to determine TG activity as previously described.23 (link) Intact tissues were incubated with 0.1 mmol/L of 5‐(biotinamido)pentylamine (BPA) and 1 mmol/L Ca2+ at 37°C for 4 hours in culture media (phenol red‐free DMEM supplemented with 2% FBS and penicillin/streptomycin) and then rinsed to free it of unreacted BPA using PBS. Samples were then homogenized in 1× RIPA buffer containing protease and phosphatase inhibitors. Proteins (0.5 to 1 μg) were loaded onto nitrocellulose membrane (BioDot Dot Bolt apparatus; Bio‐Rad). The membrane was rinsed and blocked in 3% BSA overnight and probed with HRP‐conjugated streptavidin (1:10 000 dilution in 3% BSA; Amersham Bioscience, Foster City, CA) to determine BPA incorporation. Blots were stripped using Restore Plus stripping buffer (Pierce) and reprobed with GAPDH to determine protein loading. BPA incorporation and GAPDH levels were determined by densitometry analysis using ImageJ software. For each sample, activity was calculated as the ratio of BPA/GAPDH. The nonspecific TG inhibitor, cystamine, was from Sigma‐Aldrich (St. Louis, MO), and the nonpeptidyl active site‐directed specific TG2/Factor XIII inhibitor, L682.777, was from Zedira (Darmstadt, Germany).
4
Quantifying Chicken IFNγ by ELISA
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Supernatants from the stimulated splenocytes were harvested and examined by sandwich ELISA. Briefly, anti-chicken IFNγ (2 μg/ml, Invitrogen) was coated onto 96-well maxisorp ELISA plates (Thermo Fisher Scientific). The coated plates were blocked at room temperature with PBS containing 3% BSA for 1 h. A 1:2 dilution of the supernatants was made in PBS buffer containing 3% BSA. The plates were then incubated with the diluted supernatants for 2 h at room temperature. Detection was carried out using biotinylated anti-chicken IFNγ detection antibody (1 μg/ml, Invitrogen) for 1 h at room temperature followed by HRP conjugated streptavidin (1:1000 dilution, Amersham) for another 1 h at room temperature. A 100 μl of Tetramethylbenzidine (TMB) substrate (BD biosciences) was added for 10 min. The reaction was stopped using 2 M H2SO4 and read at wavelength 450 nm in ELx808 Absorbance Microplate Reader (BioTek).
5
Quantifying Complement Activation
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Complement activation was monitored as the generation of C3a and sC5b-9 complexes in plasma by enzyme immunoassays (EIAs) as described previously. 26 In the C3a assay the monoclonal antibody 4SD17.3 was used for capture and biotinylated polyclonal rabbit anti-C3a and HRP-conjugated streptavidin (Amersham, Little Chalfont, UK) for detection. 26 In the sC5b-9 assay, anti neoC9 monoclonal antibody aE11 (Diatec AS, Oslo, Norway) was used for capture and polyclonal rabbit anti-C5 antibodies and HRP-conjugated anti-rabbit immunoglobulin (Dako A/S, Glostrup, Denmark) for detection. 27 Zymosan-activated serum served as standard and values are given as ng mL -1 (C3a) or arbitrary units per ml (AU mL -1 ; sC5b-9).
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