The competitive binding assay based on fluorescence polarization was performed as described previously for PAO1.18 (link) Briefly, 20 μL of a stock solution of LecBPA14 (150 nM) and fluorescent reporter ligand N-(fluorescein-5-yl)-N′-(α-l -fucopyranosyl ethylen)-thiocarbamide (15 nM) in TBS/Ca (20 mM Tris, 137 mM NaCl, 2.6 mM KCl at pH 7.4 supplemented with 1 mM CaCl2) were mixed with 10 μL serial dilutions (1 mM to 12.8 nM) of testing compounds in TBS/Ca in triplicates in black 384-well microtiter plates (Greiner Bio-One, Germany, cat no 781900). After addition of the reagents, the plate was incubated for 8–22 h at r.t. in a humidity chamber. Fluorescence emission parallel and perpendicular to the excitation plane was measured on a PheraStar FS (BMG Labtech, Germany) plate reader with excitation filters at 485 nm and emission filters at 535 nm. The measured intensities were reduced by buffer values and fluorescence polarization was calculated. The data were analyzed using BMG Labtech MARS software and/or with Graphpad Prism and fitted according to the four parameter variable slope model. Bottom and top plateaus were defined by the standard compounds l -fucose (1 ) and methyl α-d -mannoside (5 ) respectively and the data was reanalyzed with these values fixed. A minimum of three independent measurements of triplicates each was performed for every ligand.
Black 384 well microtiter plates
Manufactured by Greiner
Sourced in Germany
The Black 384-well microtiter plates are a type of laboratory equipment used for various assays and experiments. These plates have 384 individual wells, each designed to hold a small volume of liquid sample. The black color of the plates helps reduce background fluorescence and light absorption, which can be beneficial in certain types of assays. The plates are typically made of polystyrene or a similar material and are available in standard sizes and formats.
Automatically generated - may contain errors
4 protocols using black 384 well microtiter plates
1
Competitive Binding Assay for LecB
2
Fluorescence Polarization Assay for LecB Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
According
to previously described
procedures,23 (link),36 (link) 20 μL of a stock solution
of LecBPAO1 (225 nM) or LecBPA14(150 nM) and
fluorescent reporter ligand N-(fluorescein-5-yl)-N′-(α-l -fucopyranosyl ethylen)thiocarbamide
(15 nM) in TBS/Ca (20 mM Tris, 137 mM NaCl, 2.6 mM KCl at pH 7.4 supplemented
with 1 mM CaCl2) were mixed with 10 μL of serial
dilutions (1 mM to 12.8 nM) of testing compounds in TBS/Ca in triplicates
in black 384-well microtiter plates (Greiner Bio-One, Germany, catalogue
no. 781900). After incubation at rt in a humidity chamber for 22–24
h, fluorescence emission parallel and perpendicular to the excitation
plane was measured on a PheraStar FS (BMG Labtech, Germany) plate
reader (excitation filters, 485 nm; emission filters, 535 nm). The
data were analyzed using BMG Labtech MARS software. After reduction
of the measured intensities by buffer values, the fluorescence polarization
was calculated and fitted according to the four parameter variable
slope model. Bottom and top plateaus were defined by the standard
compoundsl -fucose (39 ) and methyl α-d -mannoside (1 ), respectively, and the data was
reanalyzed with these values fixed. A minimum of three independent
measurements of triplicates each was performed for every ligand.
to previously described
procedures,23 (link),36 (link) 20 μL of a stock solution
of LecBPAO1 (225 nM) or LecBPA14(150 nM) and
fluorescent reporter ligand N-(fluorescein-5-yl)-N′-(α-
(15 nM) in TBS/Ca (20 mM Tris, 137 mM NaCl, 2.6 mM KCl at pH 7.4 supplemented
with 1 mM CaCl2) were mixed with 10 μL of serial
dilutions (1 mM to 12.8 nM) of testing compounds in TBS/Ca in triplicates
in black 384-well microtiter plates (Greiner Bio-One, Germany, catalogue
no. 781900). After incubation at rt in a humidity chamber for 22–24
h, fluorescence emission parallel and perpendicular to the excitation
plane was measured on a PheraStar FS (BMG Labtech, Germany) plate
reader (excitation filters, 485 nm; emission filters, 535 nm). The
data were analyzed using BMG Labtech MARS software. After reduction
of the measured intensities by buffer values, the fluorescence polarization
was calculated and fitted according to the four parameter variable
slope model. Bottom and top plateaus were defined by the standard
compounds
reanalyzed with these values fixed. A minimum of three independent
measurements of triplicates each was performed for every ligand.
3
Fluorescence-Based Lectin Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A serial dilution of the test compounds was prepared in
TBS/Ca (8.0 g/L NaCl, 2.4 g/L Tris, 0.19 g/L KCl, 0.15 g/L CaCl2·2H2O), with 30% DMSO as a co-solvent. A concentrated
solution of LecA was diluted in TBS/Ca together with the fluorescent
reporter ligand (N-(fluorescein-5-yl)-N′-(β-d -(m-aminophenyl)galactopyranosyl)thiocarbamide)
to yield concentrations of 40 μM and 20 nM, respectively. A
10 μL solution of this mix was added to 10 μL serial dilutions
of the test compounds in a black 384-well microtiter plates (Greiner
Bio-One, Germany, cat. no. 781900) in triplicate. After centrifugation
(2680 rcf, 1 min, r.t.), the reactions were incubated for 30–60
min at r.t. in a humidity chamber. Fluorescence (excitation 485 nm,
emission 535 nm) was measured in parallel and perpendicular to the
excitation plane on a PheraStar FS plate reader (BMG Labtech GmbH,
Germany). The measured intensities were reduced by the values of only
LecA in TBS/Ca, and fluorescence polarization was calculated. The
data were analyzed with the MARS Data Analysis Software (BMG Labtech
GmbH, Germany) and fitted according to the four-parameter variable
slope model. Bottom and top plateaus were fixed according to the control
compounds in each assay (p-nitrophenyl)-β-d -galactoside), and the data was reanalyzed with these values
fixed. A minimum of three independent measurements on three plates
was performed for each inhibitor.
TBS/Ca (8.0 g/L NaCl, 2.4 g/L Tris, 0.19 g/L KCl, 0.15 g/L CaCl2·2H2O), with 30% DMSO as a co-solvent. A concentrated
solution of LecA was diluted in TBS/Ca together with the fluorescent
reporter ligand (N-(fluorescein-5-yl)-N′-(β-
to yield concentrations of 40 μM and 20 nM, respectively. A
10 μL solution of this mix was added to 10 μL serial dilutions
of the test compounds in a black 384-well microtiter plates (Greiner
Bio-One, Germany, cat. no. 781900) in triplicate. After centrifugation
(2680 rcf, 1 min, r.t.), the reactions were incubated for 30–60
min at r.t. in a humidity chamber. Fluorescence (excitation 485 nm,
emission 535 nm) was measured in parallel and perpendicular to the
excitation plane on a PheraStar FS plate reader (BMG Labtech GmbH,
Germany). The measured intensities were reduced by the values of only
LecA in TBS/Ca, and fluorescence polarization was calculated. The
data were analyzed with the MARS Data Analysis Software (BMG Labtech
GmbH, Germany) and fitted according to the four-parameter variable
slope model. Bottom and top plateaus were fixed according to the control
compounds in each assay (p-nitrophenyl)-β-
fixed. A minimum of three independent measurements on three plates
was performed for each inhibitor.
4
LecB Fluorescence-based Inhibition Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A serial dilution of
the test compounds was prepared in TBS/Ca, with 10% DMSO as a co-solvent.
A concentrated solution of LecB PAO1 or PA14 was diluted in TBS/Ca
together with the fluorescent reporter ligand (N-(fluorescein-5-yl)-N′-(α-l -fucopyranosyl ethylene)thiocarbamide)
to yield concentrations of 300 nM and 20 nM, respectively. A 10 μL
solution of this mix was added to 10 μL serial dilutions of
the test compounds in a black 384-well microtiter plates (Greiner
Bio-One, Germany, cat. no. 781900) in triplicate. After centrifugation
(2680 rcf, 1 min, r.t.), the reactions were incubated for 4–8
h at r.t. in a humidity chamber. Fluorescence was measured and analyzed
as for LecA. Bottom and top plateaus were fixed according to the control
compound in each assay (l -fucose), and the data were reanalyzed
with these values fixed. A minimum of three independent measurements
on three plates was performed for each inhibitor.
the test compounds was prepared in TBS/Ca, with 10% DMSO as a co-solvent.
A concentrated solution of LecB PAO1 or PA14 was diluted in TBS/Ca
together with the fluorescent reporter ligand (N-(fluorescein-5-yl)-N′-(α-
to yield concentrations of 300 nM and 20 nM, respectively. A 10 μL
solution of this mix was added to 10 μL serial dilutions of
the test compounds in a black 384-well microtiter plates (Greiner
Bio-One, Germany, cat. no. 781900) in triplicate. After centrifugation
(2680 rcf, 1 min, r.t.), the reactions were incubated for 4–8
h at r.t. in a humidity chamber. Fluorescence was measured and analyzed
as for LecA. Bottom and top plateaus were fixed according to the control
compound in each assay (
with these values fixed. A minimum of three independent measurements
on three plates was performed for each inhibitor.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!