P 20 surfactant

Manufactured by GE Healthcare

Sourced in United States, United Kingdom

The P-20 surfactant is a laboratory reagent used to facilitate the dispersion and mixing of samples in various analytical procedures. It functions as a wetting agent, reducing the surface tension of liquids and allowing for improved sample handling and preparation.

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Lab products found in correlation

Acetylated bsa, by Promega (3 mentions) Biacore t200, by Cytiva (3 mentions) Bovine serum albumin (bsa), by GE Healthcare (2 mentions) Syto59, by Thermo Fisher Scientific (1 mentions) Adenosine triphosphate (atp), by Roche (1 mentions) Biacore t100, by GE Healthcare (1 mentions) Sa chip, by GE Healthcare (1 mentions) Cm5 chip surface, by GE Healthcare (1 mentions) T100 instrument, by Cytiva (1 mentions) Ethanolamine, by GE Healthcare (1 mentions) Flag m2 antibody, by Merck Group (1 mentions) Biacore cm5 sensor chips, by Cytiva (1 mentions) Biaevaluation 4, by GE Healthcare (1 mentions) Biacore t200, by GE Healthcare (1 mentions)

12 protocols using p 20 surfactant

Single-Molecule TIRF Imaging of EcMutS

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Single-molecule fluorescence data were attained on a home-built prism-type TIRF microscope (26 (link)) with green (532 nm) and red (635 nm) laser lines. Emissions were split by a Dual View optical setup (DV2, Photometrics) in bypass mode before collection using an EMCCD camera (ProEM Exelon512, Princeton Instruments). Laser excitation was modulated by the opening and closing of shutter controlled by Micro-Manager image capture software (78 ).
The single-molecule imaging buffer was 20 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 0.1 mM DTT, 200 μg/ml acetylated BSA (Promega), 0.0025% P-20 surfactant (GE Healthcare), 1 mM ATP (Roche), and stated [NaCl]. The imaging buffer included saturated (∼3 mM) Trolox, PCA (1 mM), and PCD (10 nM) to minimize photo blinking and photobleaching (79 (link), 80 (link)).
EcMutS lifetime was calculated by flowing AF647-EcMutS (5 nM) into the prepared flow cell. In the absence of flow protein–DNA interactions were observed live. After recording Syto 59 (1000 nM, Invitrogen) was used to stain the DNA. At least two laser power settings were examined to clearly distinguish lifetime-dependent dissociation from fluorophore photobleaching. Reported lifetimes varied less than 10% at two laser power settings.

Britton B.M., London J.A., Martin-Lopez J., Jones N.D., Liu J., Lee J.B, & Fishel R. (2022). Exploiting the distinctive properties of the bacterial and human MutS homolog sliding clamps on mismatched DNA. The Journal of Biological Chemistry, 298(11), 102505.

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IgG Fc Binding Kinetics Assay

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Mouse IgG2b Fc and IgG2c Fc were immobilized on a CM5 sensor chip using amine coupling on a Biacore T100 (GE Healthcare). Lane 1 was used for a control with no protein immobilized. Carboxymethyl dextran was activated by flowing over a 1:1 mixture of 0.4 M 1-ethyl-3(3-dimethylaminopropyl)carbodiimide and 0.1M N-hydroxy-succinimide for 7 min at 5 μL/min. Mouse IgG samples were diluted to a concentration of 1 μg/mL in 10 mM sodium acetate, pH 5.0, and flowed over the CM5 chip for 10 min at 5 μL/min. Unreacted sites were blocked with 1 M ethanolamine, pH 8.5, for 7 min. Typical immobilization response units varied from 400–1000. The CM5 chip used a coupling buffer containing 20 mM MOPS, 100 mM sodium chloride, and 0.05% P20 surfactant (GE Healthcare). The binding analysis buffer was identical but contained 1 μM bovine serum albumin. All experiments were carried out at 25°C. The CM5 chip was regenerated between runs with 100 mM glycine, pH 3.0, washes for 30 s to remove any bound receptor. Dissociation constants and rates were determined by averaging the values from at least two independent experiments. The error for this mean was determined by propagating the errors associated with the least-squares fit of each individual value. Differences in values were assessed by comparing the propagated error associated with each mean value.

Falconer D.J, & Barb A.W. (2018). Mouse IgG2c Fc loop residues promote greater receptor-binding affinity than mouse IgG2b or human IgG1. PLoS ONE, 13(2), e0192123.

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Binding Kinetics of JNJ4796 by SPR

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All SPR experiments were performed using a Biacore T200 instrument operating at 25°C. Biotinylated HA was covalently immobilized on a streptavidin-coated, carboxymethylated dextran sensor surface (SA chip, GE Healthcare). JNJ4796 was dissolved at 10mM in 100% DMSO and then diluted in the running buffer (20 mM PBS, 137 mM NaCl, 0.05% P-20 surfactant, pH 7.4 (GE Healthcare), supplemented with 2% DMSO). Binding constants were obtained from a series of injections of JNJ4796 from 0.1 nM to 1 µM with a flow rate of 30 µl/min. Data from single-cycle kinetics were analyzed using BIAevaluation software. Base lines were adjusted to zero for all curves, and injection start times were aligned. The reference sensorgrams were subtracted from the experimental sensorgrams to yield curves representing specific binding followed by background subtraction (i.e. double-referencing). Binding kinetics was evaluated using a 1:1 binding model (Langmuir) to obtain association rate constants (k_a) and dissociation rate constants (k_d). Binding affinity (K_D) was estimated from the concentration dependence of the observed steady-state responses.

van Dongen M.J., Kadam R.U., Juraszek J., Lawson E., Brandenburg B., Schmitz F., Schepens W.B., Stoops B., van Diepen H.A., Jongeneelen M., Tang C., Vermond J., Real A.V., Blokland S., Garg D., Wu W., Goutier W., Lanckacker E., Klap J.M., Peeters D.C., Wu J., Buyck C., Jonckers T.H., Roymans D., Roevens P., Vogels R., Koudstaal W., Friesen R.H., Raboisson P., Dhanak D., Goudsmit J, & Wilson I.A. (2019). An orally active small molecule fusion inhibitor of influenza virus. Science (New York, N.Y.), 363(6431), eaar6221.

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Single-Molecule Imaging Buffer Composition

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The single-molecule Imaging Buffer A contains 20 mM Tris-HCl (pH 7.5), 0.1 mM DTT, 0.2 mg/mL acetylated BSA (Molecular Cloning Laboratories), 0.0025% P-20 surfactant (GE healthcare), 1 mM ATP, 5 mM MgCl₂ (unless stated otherwise) and 100 mM NaCl (unless stated otherwise). To minimize photoblinking and photobleaching, All Imaging Buffer was supplemented with a photostability enhancing and oxygen scavenging cocktail containing saturated (~3 mM) Trolox and PCA/PCD oxygen scavenger system composed of PCA (1 mM) and PCD (10 nM)^{69 (link)}.

Yang X.W., Han X.P., Han C., London J., Fishel R, & Liu J. (2022). MutS functions as a clamp loader by positioning MutL on the DNA during mismatch repair. Nature Communications, 13, 5808.

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IgG1 Fc Immobilization and SPR Binding

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IgG1 Fc was immobilized on a CM5 chip surface (GE Life Sciences) by performing standard amine coupling procedures on a Biacore T100 instrument. The carboxymethyl dextran surface was activated by 1:1 mixture of 0.4 M 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.1 M N-hydroxy-succinimide for 7 min at a flow rate of 5 μl/min. IgG Fc was applied to the chip at 1 μg/mL in 10 mM sodium acetate, pH 5.0 buffer and at a flow rate of 5 μl/min. Residual functional sites were deactivated by washing with 1 M ethanolamine, pH 8.5 for 7 min. Final immobilization response units were between 400–700. Flow line 1 was used as a blank with no IgG1 Fc immobilized on all sensor chips. All SPR measurements were performed at 25°C. The binding analyses were performed with binding buffer containing 20 mM MOPS, 100 mM sodium chloride, 1 μM bovine serum albumin and 0.05% P20 surfactant (v/v, GE Life Sciences), pH 7.2. The CM5 chip surface was regenerated by a 100 mM glycine, pH 3.0 wash for 30 s to remove bound receptor. A minimum of one replication for each condition was collected on different days. Representative results are shown.

Falconer D.J., Subedi G.P., Marcella A.M, & Barb A.W. (2018). Antibody fucosylation lowers FcγRIIIa/CD16a affinity by limiting the conformations sampled by the N162-glycan. ACS chemical biology, 13(8), 2179-2189.

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Single-Molecule Imaging Buffer Composition

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Single-molecule imaging Buffer C contains 20 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 100 mM NaCl, 0.1 mM DTT, 0.2 mg/mL acetylated BSA (Promega) and 0.0025% P-20 surfactant (GE healthcare). To minimize photoblinking and photobleaching, imaging buffer was supplemented with a photostability enhancing and oxygen scavenging cocktail containing saturated (~3 mM) Trolox and PCA/PCD oxygen scavenger system composed of PCA (1 mM) and PCD (10 nM)^{72 (link)}.

Liu J., Lee R., Britton B.M., London J.A., Yang K., Hanne J., Lee J.B, & Fishel R. (2019). MutL sliding clamps coordinate exonuclease-independent Escherichia coli mismatch repair. Nature Communications, 10, 5294.

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Surface Plasmon Resonance Assay

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The surface plasmon resonance assays were conducted on a Biacore T200 instrument using Biacore CM5 sensor chips (Biacore). Ethanolamine, N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) and P-20 surfactant were all obtained from GE Lifesciences. M2 Flag antibody was obtained from Sigma. Reference proteins were obtained from Origene.

Pop M.S., Stransky N., Garvie C.W., Theurillat J.P., Lewis T.A., Zhong C., Culyba E.K., Lin F., Daniels D.S., Pagliarini R., Ronco L., Koehler A.N, & Garraway L.A. (2014). A small molecule that binds and inhibits the ETV1 transcription factor oncoprotein. Molecular cancer therapeutics, 13(6), 1492-1502.

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Purification and Characterization of Factor H

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Complement protein Factor H was purified from normal human plasma as previously described (31 (link)). Purified Factor H was >97% homogenous by polyacrylamide gel electrophoresis with an apparent molecular weight on SDS gel electrophoresis of 155,000 in its reduced form. Protein concentration was determined spectrophotometrically using an E_280nm (1% solution) of 12.4 for Factor H and its recombinant fragments. Phosphate-buffered saline (PBS) was 10 mM phosphate, 145 mM NaCl, 0.02% sodium azide and pH 7.4. Veronal-buffered saline (VBS) was 5 mM veronal, 145 mM NaCl, 0.02% sodium azide, and pH 7.4. Gelatin VBS (GVB) was VBS containing 0.1% gelatin, while GVBE was GVB containing 10 mM EDTA. HEPES-buffered saline (HBS-P) contained 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% P20 surfactant (GE Biosciences), and 0.02% sodium azide. All buffers were made to be similar and close to physiological isoelectric strength and the physiological pH to allow comparisons between measurements to be made.

Haque A., Cortes C., Alam M.N., Sreedhar M., Ferreira V.P, & Pangburn M.K. (2020). Characterization of Binding Properties of Individual Functional Sites of Human Complement Factor H. Frontiers in Immunology, 11, 1728.

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FcRn Binding Kinetics of Antibody Variants

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SPR experiments were performed on Bia3000 apparatus at 25 °C in 50 mM phosphate buffer with 150 mM NaCl containing 0.05% P20 surfactant (GE Healthcare, Chicago, IL, USA) adjusted at pH 7 or pH 6 as required. hFcRn (Immunitrack, Copenhagen, Denmark) was immobilized in acetate buffer at pH 5 on CM5 sensor chips at a level lower than 200 RU. Increasing concentrations of antibody variants were injected over 180 s. After a dissociation phase of 400 s, the FcRn-coated sensor chip was regenerated by a pulse of 10 mM NaOH and PBS. The multi-cycle kinetics were evaluated by a bivalent model fitting (BiaEvaluation 4.1.1, GE Healthcare). Each variant was analyzed on freshly immobilized hFcRn.

Dumet C., Pugnière M., Henriquet C., Gouilleux-Gruart V., Poupon A, & Watier H. (2023). Harnessing Fc/FcRn Affinity Data from Patents with Different Machine Learning Methods. International Journal of Molecular Sciences, 24(6), 5724.

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CD16a Affinity Measurement by Biacore

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All affinity measurements were performed with a Biacore T200 (GE Life Sciences) using amine coupling to a CM5 chip. Fc was coupled at 1 μg/ml for saturation experiments. Several different CD16a concentrations, ranging between 0.01 μM and 20.48 μM, was flowed over the chip surface at a rate of 10 μl/min. CD16a was diluted in running buffer containing 20 mM MOPS, 100 mM sodium chloride, 1 μM bovine serum albumin, and 0.05% P20 surfactant (GE Life Sciences), pH 7.4. Contact and dissociation times of at least 300 s were used for all proteins. The chip was regenerated after each step with 100 mM glycine, pH 3.0 for 30 s.

Kremer P.G, & Barb A.W. (2022). The weaker-binding Fc γ receptor IIIa F158 allotype retains sensitivity to N-glycan composition and exhibits a destabilized antibody-binding interface. The Journal of Biological Chemistry, 298(9), 102329.

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