Antibody affinity to Stx1 (E167Q) was measured using an Octet QK system (Forte-bio, Menlo Park, CA) as previously described [28] (link). Briefly, biotinylated mAbs were bound to streptavidin biosensors at 10 µg/mL, diluted in PBS. Stx1 (E167Q) was then incubated with the sensors at four different concentrations (142, 71, 36, and 18 nM) and then allowed to dissociate in PBS. Dissociation constants (KD) were calculated using the Octet QK software (Data Acquisition 7.0). Antibody isotyping was conducted by ELISA using an isotype-specific antibody panel (Southern Biotech, Birmingham, AL). Stx1 (E167Q) at 500 ng/mL and 100 µL/well was coated onto a clear Nunc Maxisorp ELISA plate overnight at 4°C. The plate was washed twice with PBST, then blocked with 5% nonfat dry milk/PBST for one hour at RT (200 µL/well). The plate was washed twice more, and 100 ng/mL mAb was added (100 µL/well) for one hour at RT. The plate was then washed six times, then HRP-conjugated isotype-specific antibody was added at a 1/1000 dilution (100 µL/mL) and the plate was incubated for one hour at RT. The plate was washed a final six times, TMB substrate was added at 100 µL/well, and after 3 minutes 100 µL/well 0.3 N HCl was added. Absorbance was then read at 450 nm on the Victor 3 plate reader.
Octet qk system
Manufactured by Molecular Devices
Sourced in United States
The Octet QK system is a label-free, real-time interaction analysis instrument. It measures biomolecular interactions, including protein-protein, protein-small molecule, and protein-lipid interactions. The system uses biolayer interferometry (BLI) technology to quantify association and dissociation kinetics, affinity, and concentration of analytes in solution.
Automatically generated - may contain errors
Lab products found in correlation
96 well plate, by Greiner (3 mentions)
Data acquisition 6, by Molecular Devices (2 mentions)
Maxisorp elisa plate, by Thermo Fisher Scientific (1 mentions)
Octet qk, by Molecular Devices (1 mentions)
Sephadex g 25, by GE Healthcare (1 mentions)
Ez link sulfo nhs lc lc biotin, by Thermo Fisher Scientific (1 mentions)
Recombinant mouse tnf alpha, by R&D Systems (1 mentions)
Streptavidin biosensor, by Molecular Devices (1 mentions)
96 well microplate, by Greiner (1 mentions)
Anti higg fc capture ahc biosensors, by Molecular Devices (1 mentions)
Ni nta biosensor, by Molecular Devices (1 mentions)
15 protocols using octet qk system
1
Affinity Measurement of Anti-Stx1 Antibodies
2
Biolayer Interferometry Protein Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The dissociation constants (Kd) were obtained with biolayer interferometry by using an Octet QK system (FortéBio). Samples or buffer were dispensed into 96-well plates (Greiner Bio-one) at 200 μL per well. Operating temperature was maintained at 30 °C. Proteins were diluted into kinetic buffer (PBS with 0.1% BSA and 0.002% Tween-20) and immobilized on either anti-GST or Ni-NTA sensor tips. The other proteins were diluted using the same buffer into a range of different concentrations. Assays with Ubc12N8 (Ubc12C111SN8-His) were carried out using the acidic kinetic buffer (50 mM NaOAc pH 5.5, 50 mM NaCl, 0.1% BSA, and 0.002% Tween-20). The raw data were processed by subtraction to reference cells and then aligned with associations (0.2-0.4s). The dissociation constants Kd were obtained by fitting the processed data using the 1:1 model in the Octet analysis software with R2 >0.99 and labeled as mean ± S.D.
3
MERS-HR2P and MERS-5HB binding affinity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Real-time binding assay between peptide MERS-HR2P and MERS-5HB was carried out on an Octet QK system (Fortebio, San Francisco, CA, USA) at 25 °C using biolayer interferometry technology. Specifically, biotin-labeled 500 ng/mL MERS-HR2P was loaded into streptavidin biosensors and then MERS-5HB was added at a series of concentrations: 64 nM, 32 nM, 16 nM, 8 nM, 4 nM, 2 nM. Protein dilutions were dissolved in PBS containing 0.02% Tween (Sangon Biotech), which is better for reducing nonspecific adsorption. Association and dissociation time were both set as 1200 s. The affinity of MERS-HR2P to MERS-5HB was (calculated automatically using the Octet QK software package) (Fortebio) by a 1:1 binding model, and the equilibrium dissociation constant (KD) value was equal to the kinetic dissociation rate constant divided by the kinetic association rate constant.
4
Kinetic Analysis of G-CSF-GCSFR Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bio-layer interferometry (BLI) was used to detect the binding of G-CSF to G-CSFR under various conditions using a Streptavidin High Binding Biosensor Kit and a Octet-QK system (Fortebio, USA). Biotinylated rhG-CSFR was desalted with Sephadex G25 (GE Healthcare, USA) and eluted in a final concentration of 15 µg/ml. Sample preparation, hydration of the sensors, and kinetic analysis of macromolecular interactions were performed according to the manufacturer's instructions. Then 200 µl of HMG, rhG-CSF standard (Amgen, USA), and mG-CSF or buffer were transferred into a 96-well plate (Greiner, Austria) and assayed for rapid assessment of kinetics. Data were analyzed using global fitting to determine the association/dissociation rate constants and affinity constant.
5
Transient IgG Expression in HEK293T
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 3
Transfection of the various plasmids encoding the recloned VH variants, and further encoding the common light chain huIGKV1-39, in HEK293T cells was performed according to standard procedures such that IgG could express (de Kruif et al Biotech Bioeng. 2010). After transfection, IgG expression levels in supernatants were measured using the ForteBIO Octet-QK system, which is based on Bio-Layer Interferometry (BLI) and which enables real-time quantitation and kinetic characterization of biomolecular interactions; for details see www.fortebio.com. When expression levels exceeding 5 μg/ml were measured, the IgG was purified using Protein A affinity purification.
6
Quantifying Monoclonal Antibody Titers by BLI
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Product titer in culture supernatants was determined by bio‐layer interferometry (BLI) with the Octet QK system (Pall Forte Bio), as previously described by Sissolak et al.16 (link) Briefly, samples were diluted in phosphate buffered saline with 0.1 vol% Tween 20 (PBS‐T) and transferred to each well of a black, nonsterile 96‐well plate (Thermo Fischer Scientific). Protein A biosensor tips were equilibrated in PBS‐T, and plates were shaken at 1000 rpm for 5 s before each measurement. The binding rates of mAb to Protein A were determined over a time interval of 300 , and mAb quantity was calculated via a calibration curve in the concentration range between 10 and 50 μg/ml. Calculation was performed using Octet software (version 6.4, ForteBio).
7
Binding Affinity of Anti-TNF-α Antibody
8
Antibody Affinity to Modified Stx2e
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibody affinity to Stx2e (E167Q) was measured using an Octet QK system (Forte-bio, Menlo Park, CA) as previously described [8 (link)]. Briefly, biotinylated mAbs were bound to streptavidin biosensors at 10 μg/mL, diluted in PBS. Stx2e (E167Q) was then incubated with the sensors at four different concentrations (142, 71, 36, and 18 nM) and then allowed to dissociate in PBS. Dissociation constants (KD) were calculated using the Octet QK software (Data Acquisition 7.0).
9
IgG Expression and Purification in HEK293T
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 3
Transfection of the various plasmids encoding the recloned VH variants, and further encoding the common light chain huIGKV1-39, in HEK293T cells was performed according to standard procedures such that IgG could express (de Kruif et al Biotech Bioeng. 2010). After transfection, IgG expression levels in supernatants were measured using the ForteBIO Octet-QK system, which is based on Bio-Layer Interferometry (BLI) and which enables real-time quantitation and kinetic characterization of biomolecular interactions; for details see www.fortebio.com. When expression levels exceeding 5 μg/ml were measured, the IgG was purified using Protein A affinity purification.
10
IgG Expression and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 3
Transfection of the various plasmids encoding the recloned VH variants, and further encoding the common light chain huIGKV1-39, in HEK293T cells was performed according to standard procedures such that IgG could express (de Kruif et al Biotech Bioeng. 2010). After transfection, IgG expression levels in supernatants were measured using the ForteBIO Octet-QK system, which is based on Bio-Layer Interferometry (BLI) and which enables real-time quantitation and kinetic characterization of biomolecular interactions; for details see www.fortebio.com. When expression levels exceeding 5 μg/ml were measured, the IgG was purified using Protein A affinity purification.
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!