Proteasomes were reconstituted at 2X concentration with limiting concentrations of core particle (100 nM final) and saturating concentrations of base, lid, and Rpn10 (2 μM final) for 5 min at room temperature in assay buffer (GF buffer supplemented with 5 mM ATP, 1 mg/mL BSA, and ATP regeneration (creatine kinase and creatine phosphate)). Fluorescein labeled titin-I27 with a V15P mutation and a C-terminal 35 residue tail (FAM-titin-I27V15P) was prepared at 2X final concentration in assay buffer. Reactions were initiated with 5 μL of proteasome sample being added to 5 μL of FAM-titin-I27V15P substrate in a 384-well flat bottom black corning plate. Degradation was monitored by the loss of fluorescence anisotropy of conjugated fluorescein over time in a Synergy Neo2 Multi-Mode Plate Reader (Biotek). Degradation rates were calculated by determining the fluorescence anisotropy difference between substrate and substrate peptides and applying linear regression to initial anisotropy decreases. Initial rates were plotted against substrate concentration and fitted to the Michaelis-Menten equation (OriginPro9) to determine kcat and Km values.
Synergy neo2 multi mode plate reader
Manufactured by Agilent Technologies
The Synergy Neo2 Multi-Mode Plate Reader is a versatile laboratory instrument designed for high-performance detection in a wide range of applications. It offers multi-mode detection capabilities, including absorbance, fluorescence, and luminescence, allowing users to conduct diverse assays within a single platform.
Automatically generated - may contain errors
Lab products found in correlation
Amikacin ami, by Chem-Impex (2 mentions)
Tobramycin tob, by Thermo Fisher Scientific (2 mentions)
Cefepime cef, by Chem-Impex (2 mentions)
Ciprofloxacin cip, by Enzo Life Sciences (2 mentions)
Imipenem imi, by Combi-Blocks (2 mentions)
Gentamicin gen, by Merck Group (2 mentions)
Vancomycin van, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
96 well plate, by Corning (1 mentions)
Aposensor cell viability assay kit, by Abcam (1 mentions)
Texas red dextran, by Merck Group (1 mentions)
Matlab, by MathWorks (1 mentions)
Eu anti gst antibody, by Thermo Fisher Scientific (1 mentions)
Proxiplate white 384 well plates, by PerkinElmer (1 mentions)
Eu streptavidin, by Thermo Fisher Scientific (1 mentions)
Prism 9, by GraphPad (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Infinite m plex plate reader, by Tecan (1 mentions)
Infinite plate reader, by Tecan (1 mentions)
Chymotrypsin, by Merck Group (1 mentions)
21 protocols using synergy neo2 multi mode plate reader
1
Proteasome Activity Assay with Fluorescent Titin
2
Reconstitution and Kinetics of Proteasomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteasomes were reconstituted at 2X final concentration with limiting concentration of core particle (10 nM final) and saturating concentrations of base (0.5 μM (1X) or 1 μM (2X) for experiments where the base concentration was doubled), lid (2 μM), and Rpn10 (2 μM) in GF buffer supplemented with 0.5 mM TCEP and 5 mM ATP for 5 min at room temperature. Reconstituted proteasomes were incubated in either 5 mM ATP or 5 mM ATPγS at room temperature for an additional 5 min. Suc-LLVY-AMC was diluted to 2X concentration (100 μM final) in 26S GF buffer. Reactions were initiated by aliquoting 5 μL of reconstituted proteasomes into 5 μL of Suc-LLVY-AMC solution in a 384-well flat bottom black corning plate. Suc-LLVY-AMC hydrolysis was tracked by the increase in fluorescence upon AMC release in a Synergy Neo2 Multi-Mode Plate Reader (Biotek). Data were fit by linear regression, and slopes were normalized to wild-type proteasomes in ATP.
3
Preosteoblast MC3T3-E1 Cell Culture and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Preosteoblast MC3T3-E1 cells (CRL-2593; ATCC, Manassas, VA) were cultured as described previously25 (link). Briefly, cells were cultured in complete alpha minimal essential media (α-MEM) containing 10% fetal bovine serum (FBS) (Invitrogen and Atlanta Biologicals, Atlanta, GA) and 1% Penicillin-Streptomycin (Life Technologies). Passages between 18 and 24 were used for the experiments. For gene expression analysis, cells were plated in 96-well plates (@ 20,000 cells/well) (Corning Incorporated, Corning NY). Following 24 hours of plating in complete α-MEM, cells were treated as indicated for 6 hours. RNA extraction was completed as described below.
For assessment of intracellular ATP, MC3T3-E1 cells were plated @10,000 cells/well in 96-well plates (white-walled from Corning Incorporated, Corning NY). Twenty four hours after plating, cells were treated as indicated for 6 hours. Intracellular ATP levels were assessed using the ApoSensor Cell Viability Assay kit (BioVision, San Francisco, CA) and Luminescence was measured by Synergy/neo2 multi-mode plate reader and calculated with Gen5 software (Bio-Tek). Each experiment was done in duplicates.
For assessment of intracellular ATP, MC3T3-E1 cells were plated @10,000 cells/well in 96-well plates (white-walled from Corning Incorporated, Corning NY). Twenty four hours after plating, cells were treated as indicated for 6 hours. Intracellular ATP levels were assessed using the ApoSensor Cell Viability Assay kit (BioVision, San Francisco, CA) and Luminescence was measured by Synergy/neo2 multi-mode plate reader and calculated with Gen5 software (Bio-Tek). Each experiment was done in duplicates.
4
DPPH Antioxidant Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the anti-oxidative activity of 3-HT and CDA, the DPPH free radical scavenging method was used as described previously [43 (link)]. DPPH was dissolved in 99% ethanol and kept in the dark for 2 h. DPPH solution (1 mL) was added to a 24-well plate, followed by 200 µL of ethanol, 3-HT, or CDA, and 800 µL of 0.1 M Tris-HCl buffer (pH 7.4). After 30 min of incubation in the dark, absorbance was measured at 517 nm using the Synergy™ Neo2 Multi-Mode plate reader (BioTek).
5
Measuring Epithelial Permeability with Fluorescent Dextrans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Epithelial permeability across polarized 16HBE14o- monolayers was assessed by measuring the flux of fluorescein isothiocyanate- (FITC)-label dextran (molecular weight 4 kDa, Sigma-Aldrich) and Texas-red-dextran (molecular weight 10 kDa, Sigma-Aldrich) from the apical to basolateral compartments. At 48 h after treatment, the upper and lower compartments of treated media were aspirated. Thereafter, serum-free media with either 1 mg/mL FITC- or 50 μg/mL Texas-red-conjugated dextran was directly added into the apical compartment. Paracellular flux was assessed by taking 100 μL aliquots from the basolateral compartment after 2 hours of sterile incubation at 37°C. The amount of fluorophore that diffused into the lower chamber was measured by the fluorescent microplate reader (Synergy™ Neo2 multimode plate reader, BioTek Instruments) at an excitation and emission wavelength of 485 nm and 535 nm, respectively. The FITC- and Texas-red-conjugated dextran concentration was calculated with a known standard concentration curve.
6
Reversible Tyrosine and Serine Switches
7
Ubp6 Activity Measurement Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ubp6 activity was measured using the cleavage of the fluorogenic Ub-AMC substrate (Life Sensors). Proteasomes were reconstituted as base-limited complexes (at a final concentration of 200 nM base, 1.2 µM lid, 600 µM core, 1.5 µM Rpn10∆UIM) in either 1X ATP regeneration mix or 4 mM ATPγS with 40 nM Ubp6. After 4 min preincubation at 25 °C, samples were mixed with Ub-AMC to a final concentration of 10 µM. Cleavage was measured by monitoring the change of fluorescence at 445 nm after excitation at 345 nm on a plate reader (Synergy Neo2 Multi-Mode Plate Reader, Biotek).
8
Kinase Binding Assay for RSK2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The relative amount of inhibitor bound to RSK2 was determined using the LanthaScreen Eu Kinase Binding Assay for RPS6KA3 (Invitrogen). Kinase, Eu anti-His antibody, inhibitor or vehicle, and Kinase Tracer 236 (tracer) in kinase buffer were incubated as described in Figure 1(D) , with final assay concentrations of 5 nM kinase, 2 nM antibody, inhibitor or vehicle (0.95% DMSO), and 15 nM tracer in Proxiplate white 384-well plates (Perkin Elmer) with 15 μL total assay volume per well. For RSK1 and RSK2 NTKD, 2 nM Eu Streptavidin and 2 nM Biotin anti-His antibody (Invitrogen) and for RSK3/4, 2 nM Eu anti-GST antibody (Invitrogen) were used in place of the Eu anti-His antibody. Time resolved fluorescence was measured with a Synergy Neo 2 Multi Mode Plate Reader and Gen5 software (Biotek) with excitation fluorescence at 330 ± 80 nm and emission measured at 620 ± 10 nm (background fluorescence from Eu antibody) and at 665 ± 8 nm (emission from fluorescence resonance energy transfer, FRET, from tracer) with 100 μs delay and 200 μs collection time. For each time point, emission ratio (EMR) was calculated as emission at 665 nm divided by emission at 620 nm. The corresponding average EMR of wells without inhibitor or kinase was subtracted, and background-subtracted EMR was normalised to average vehicle control. Normalised EMR was calculated in MATLAB (Mathworks).
9
Cellular ATP Quantification Assay
10
Fluorescence and Luminescence Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SfGFP fluorescence in all our studies was measured using a TECAN infinite MPLEX plate reader with excitation/emission wavelength set to 485/530 nm and gain set to 100. Luminescence imaging was quantified using a BioTek Synergy Neo2 Multi-Mode Plate Reader with gain set to 135.
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!