Microtiter plates were coated with 1 μg of each LigA or LigB fragment overnight at 4°C in PBS (130 mM NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4). After blocking with 3% BSA, C4BP (1μM—0.0156 μM, 2 fold serial dilution) was then added to the plates for 1 h at 37°C. Between each step, plates were washed with PBS-T three times. Subsequently, mouse monoclonal anti-C4BP antibodies (1:2000) (EMD Millipore) recognizing C4BP α- and β-chains were used as primary antibodies. Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibody (1:2000) (Invitrogen) was used as secondary antibody. After washing three times with PBS-T, 100 μl of 0.2 mg/ml 3,3’,5,5’-tetramethylbenzidine (TMB) substrate (Kirkegaard & Perry Laboratories) was added to each well. Finally, after a 10 min-incubation the microtiter plates were read at 630 nm using an ELISA plate reader (Biotek EL-312). Each value represents the mean ± SE of three independent experiments, each performed in triplicate.
El 312
Manufactured by Agilent Technologies
Sourced in United States
The EL-312 is a precision power supply designed for laboratory and research applications. It provides stable and adjustable direct current and voltage outputs. The core function of the EL-312 is to supply power to various electronic devices and test setups.
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Lab products found in correlation
4 protocols using el 312
1
Quantification of C4BP Binding to LigA/B
2
Cytotoxicity Assessment of 5-FU Nanoparticles
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The cytotoxicity of free 5-FU (0.01 µg/mL), MF-fabricated NPs based on PLGA with concentrations of 0.1% and 0.2% (w/v) containing the drug and control (the cells feeding with culture medium), was determined by MTT assay. To assess the relative cell viability of the samples, the cell lines were seeded on into a 96-well culture plate with a density of 5×103 cells/well. After 24 h, 48 h and 72 h of incubation, the amounts of formazan representing the alive cells were measured using an ELISA reader (EL-312, BioTek Instruments Inc., Winooski, VT, USA) at 540 nm absorbance. Afterward, the relative cell viability was obtained by dividing the optical density (OD) of each sample (recording changes in absorbance at 540 nm and a reference wavelength of 620 nm) by OD of control based on Equation (3) [37 (link)]:
3
Quantifying LigB-Fibrinogen Interactions
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The ability of LigB proteins to
bind to human plasma fibrinogen (Fg) (obtained from Sigma-Aldrich)
was assessed using an ELISA assay as previously described.20 (link) Various concentrations (0, 0.31, 0.63, 1.25,
2.5, 5, 10, and 20 μM) of histidine-tagged LigBCen2R (positive
control), LigB-4 (negative control), and LigB-12 were added to microtiter
wells coated with 1 μM Fg or BSA (negative control, data not
shown) in PBS buffer. Following 1 h incubation at 37 °C, the
wells were washed with PBS buffer containing 0.05% Tween 20 (PBS-T)
to remove unbound LigB proteins. To detect the interaction of each
LigB truncate with Fg, mouse anti-His tag antibody (1:500) and horseradish
peroxidase (HRP) conjugated goat anti-mouse IgG antibody were used
as primary and secondary antibody (Eugene). Finally, 100 μL
of HRP substrates was applied to develop the color and then the plates
were read at 630 nm with an ELISA plate reader (Biotek EL-312, Winooski,
VT). To determine the end-point dissociation constant (KD), the binding curves were fit with the following equation
using KaleidaGraph software (Abelbeck software, Reading, PA):
bind to human plasma fibrinogen (Fg) (obtained from Sigma-Aldrich)
was assessed using an ELISA assay as previously described.20 (link) Various concentrations (0, 0.31, 0.63, 1.25,
2.5, 5, 10, and 20 μM) of histidine-tagged LigBCen2R (positive
control), LigB-4 (negative control), and LigB-12 were added to microtiter
wells coated with 1 μM Fg or BSA (negative control, data not
shown) in PBS buffer. Following 1 h incubation at 37 °C, the
wells were washed with PBS buffer containing 0.05% Tween 20 (PBS-T)
to remove unbound LigB proteins. To detect the interaction of each
LigB truncate with Fg, mouse anti-His tag antibody (1:500) and horseradish
peroxidase (HRP) conjugated goat anti-mouse IgG antibody were used
as primary and secondary antibody (Eugene). Finally, 100 μL
of HRP substrates was applied to develop the color and then the plates
were read at 630 nm with an ELISA plate reader (Biotek EL-312, Winooski,
VT). To determine the end-point dissociation constant (KD), the binding curves were fit with the following equation
using KaleidaGraph software (Abelbeck software, Reading, PA):
4
Retrospective Evaluation of P. vivax Antibodies
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Antibodies against the P. vivax blood stage can last for months or years in exposed patients. However, antibody titres fade out more rapidly in treated patients [29 (link)–31 (link)]. To reveal P. vivax re-exposure, with and without clinical symptoms and/or low parasitaemia in recurrent blood infections, retrospective research was conducted at the end of the 12-month follow-up period to look for native P. vivax blood-stage proteins in preserved blood samples by using ELISA, as previously reported [32 (link)]. Briefly, the blood samples that had been taken from each patient and smeared on filter paper were eluted in PBS and tested at 1:500 dilutions in an indirect ELISA. The reaction was revealed using goat anti-human IgG (H + L)-HRP (Pierce, Rockford, IL, USA) diluted 1:5000 and ABTS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) as substrate for 60 min, and then OD values were recorded in a spectrophotometer (Biotek® model EL312) at 405 nm. Cut-off values were previously determined using unexposed individuals as the mean and 2SD as 0.25 OD values (95 % confidence). The ELISA-OD values were plotted according to time point per patient.
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