Tris glycine native running buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States

Tris-Glycine Native Running Buffer is a buffer solution used for the electrophoretic separation of proteins under non-denaturing conditions. It maintains the native structure and biological activity of proteins during the separation process.

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Lab products found in correlation

Tris glycine native sample buffer, by Thermo Fisher Scientific (4 mentions) Novex tris glycine 4 20 gels, by Thermo Fisher Scientific (2 mentions) Goat anti mouse igg hrp, by Thermo Fisher Scientific (1 mentions) N dodecyl β d maltoside ddm, by Merck Group (1 mentions) Nupage tris acetate gel, by Thermo Fisher Scientific (1 mentions) Chemidoc xrs imaging system, by Bio-Rad (1 mentions) Horseradish peroxidase labeled anti mouse antibody, by Thermo Fisher Scientific (1 mentions) Esterase from porcine liver, by Merck Group (1 mentions) Novex tris glycine gel, by Thermo Fisher Scientific (1 mentions) Seeblue pre stained protein standard, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Tris–Glycine Native buffer Tris-Glycine running buffer Native running buffer Tris-Glycine buffer Running Buffer Tris buffer Glycine

7 protocols using tris glycine native running buffer

Simultaneous Ligand Binding Assay

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Example 6

Purified PDGFRα_D123-PAS(200)-VEGFR1_D2/R2_D3, referred to herein as EPS1108P (25 pmol) was incubated with either 25 pmol VEGF-A₁₆₅(#8065-LF; Cell Signaling Technology, Danvers Mass., USA) or 25 pmol PDGF-AA (#8913-LF; Cell Signaling Technology) or both ligands (each 25 pmol), as indicated in FIG. 5, in 25 μl reactions in the presence of 20 mM HEPES/NaOH, pH 7.4, 100 mM NaCl for 30 min on ice. The solutions were then mixed with 10× native sample buffer (60 mM Tris base, 480 mM glycine, pH 8.3; 50% (v/v) glycerol, 0.01% (w/v) bromophenol blue) and immediately loaded onto a 3-8% Tris-acetate polyacrylamide gel (without SDS; Invitrogen, Carlsbad, Calif., USA). The gel was run at 90 V in Tris-glycine native running buffer, pH 8.3 (Invitrogen) at RT until the bromophenol blue marker reached the bottom of the gel. The gel was shortly rinsed in water and then stained using InstantBlue colloidal Coomassie blue protein stain (Expedeon, Cambridge, UK). The gel was documented by digital imaging. Under native conditions used for PAGE, both ligands, VEGF-A165 and PDGF-AA, bind to PDGFRαD123-PAS(200)-VEGFR1D2/R2D3 and result in stable complexes (ref. FIG. 2), that can be detected: (I) Simultaneous binding of both ligands, when both ligands are present or (II) binding of either PDGF-AA or VEGF-A165, in the absence of the other ligand (FIG. 5).

US11548931B2. PASylated VEGFR/PDGFR fusion proteins and their use in therapy (2023-01-10). XL-PROTEIN GMBH [DE]. Inventors: Qing Dong [US], Michaela Gebauer [DE].

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HCMV gB Protein Analysis by Electrophoresis

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Purified proteins were analyzed by electrophoresis on 3–8% NuPAGE Tris-Acetate Mini-Gels, under both reducing condition and modified non-reducing conditions as previously described [64 (link)]. For reducing conditions, purified HCMV gB was boiled for 10 min in lithium dodecyl sulfate (LDS) loading buffer containing 50 mM DTT, resolved on 3–8% PAGE in SDS running buffer, and blotted with an anti-gB monoclonal antibody 2F12. For modified non-reducing conditions, protein samples were mixed with LDS loading buffer without DTT, resolved on 3–8% PAGE in Tris-glycine native running buffer (Invitrogen), and blotted with anti-gB monoclonal antibody LS-C64457. Membranes were then incubated with polyclonal HRP-goat anti-mouse IgG (Thermo Fisher Scientific), followed by incubation with SuperSignal West Pico chemiluminescent substrate, with signal captured on X-ray film.

Cui X., Cao Z., Wang S., Lee R.B., Wang X., Murata H., Adler S.P., McVoy M.A, & Snapper C.M. (2018). Novel trimeric human cytomegalovirus glycoprotein B elicits a high-titer neutralizing antibody response ,. Vaccine, 36(37), 5580-5590.

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Heme Transfer Assay for PGRMC2

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Heme transfer was assessed by mixing 10 μg of WT or mouse PGRMC2 heme-binding mutant (3xM) with 10 μg of apo-REV-ERBα protein and incubating for 30 min at 37°C. After incubation, 2X Native Tris-Glycine sample buffer (Life Technologies) was added and samples separated by electrophoresis using Novex Tris-Glycine 4-20% gels and Tris-Glycine Native Running Buffer (Life Technologies) for 6 hr. The gel was washed for 10 min with water and heme staining was performed using the BioFX TMB One Component HRP Microwell Substrate (Surmodics). After imaging the heme stain, the gel was washed overnight with water and counterstained with Coomassie for protein detection.

Galmozzi A., Kok B.P., Kim A.S., Montenegro-Burke J.R., Lee J.Y., Spreafico R., Mosure S., Albert V., Cintron-Colon R., Godio C., Webb W.R., Conti B., Solt L.A., Kojetin D., Parker C.G., Peluso J.J., Pru J.K., Siuzdak G., Cravatt B.F, & Saez E. (2019). PGRMC2 is an Intracellular Heme Chaperone Critical for Adipocyte Function. Nature, 576(7785), 138-142.

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Heme Transfer Assay for PGRMC2

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BN-PAGE Gel Shift Assay for Env Variants

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BN-PAGE gel mobility shift assays were performed using virions produced by transfection of the infectious molecular clone plasmid pLAI displaying the Env variant ADA.CM (Leaman and Zwick, 2013 (link)). Virions and a dilution series of Fab were incubated at RT for 30 min. The virion/Ab mixture was solubilized in 1% n-Dodecyl β-D-maltoside (DDM; Sigma) for 20 min on ice, and then run on a 3–8% gradient Tris-Acetate NuPAGE gel (ThermoFisher) in Tris-Glycine Native Sample Buffer (ThermoFisher), supplemented with 0.25% Coomassie G-250. Gels were run for 3 h at 150 V in Tris-Glycine Native Running Buffer (ThermoFisher) + 0.002% Coomassie G-250. Proteins were transferred onto PDVF membrane and Western blotted using a cocktail of primary antibodies to gp120 (F105, 2G12 and HGN194, 2 μg/mL each) and gp41 (10E8, 2F5, and 7B2, 1 μg/mL each) followed by a goat anti-human-Fcγ-HRP secondary antibody (Jackson). The blot was developed using ECL Plus Substrate (Pierce) and a ChemiDoc XRS + Imaging System (Bio-Rad).

Rujas E., Leaman D.P., Insausti S., Carravilla P., García-Porras M., Largo E., Morillo I., Sánchez-Eugenia R., Zhang L., Cui H., Iloro I., Elortza F., Julien J.P., Eggeling C., Zwick M.B., Caaveiro J.M, & Nieva J.L. (2021). Focal accumulation of aromaticity at the CDRH3 loop mitigates 4E10 polyreactivity without altering its HIV neutralization profile. iScience, 24(9), 102987.

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Evaluation of HCMV gH/gL Protein Structure

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Purified proteins were analyzed by electrophoresis on 3–8% NuPAGE Tris-Acetate Mini-Gels under reducing conditions or modified non-reducing conditions. Under reducing conditions, purified HCMV monomeric or trimeric gH/gL recombinant proteins were boiled for 10 min in lithium dodecyl sulfate (LDS) loading buffer containing 50 mM of dithiothreitol (DTT), and resolved on 3–8% PAGE in SDS running buffer. For modified non-reducing conditions, protein samples were mixed with LDS loading buffer without DTT, and resolved on 3–8% PAGE in tris-glycine native running buffer (Thermo Fisher Scientific, Waltham, WA, USA). Proteins were transferred to membranes, and probed using a mouse monoclonal anti-gH antibody (0861) from Santa Cruz Biotechnology (Dallas, TX, USA), followed by horseradish peroxidase labeled anti-mouse antibody from Thermo Fisher Scientific (Waltham, MA, USA). Membranes were then incubated with SuperSignal West Pico chemiluminescent substrate with a signal captured on X-ray film.

Cui X., Cao Z., Wang S., Flora M., Adler S.P., McVoy M.A, & Snapper C.M. (2019). Immunization of Rabbits with Recombinant Human Cytomegalovirus Trimeric versus Monomeric gH/gL Protein Elicits Markedly Higher Titers of Antibody and Neutralization Activity. International Journal of Molecular Sciences, 20(13), 3158.

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Esterase Activity Assay in Bacterial Spores

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Proteins washed from 220 mg of spores were freeze-dried, then dissolved in 500 μL of 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1 mM EDTA, 1% v/v Triton X-100, 0.1 M PMSF, and a protease inhibitor cocktail tablet (Roche Diagnostics). Intracellular proteins were isolated from 220 mg of washed spores by grinding in liquid nitrogen. The tissue powder was dissolved in 500 μL of the same buffer as the extracellular proteins. The lysate was clarified by centrifugation (16,900 × g, 4°C, 20 min), and the supernatant was collected. Protein concentration was determined using the Bradford protein assay. Esterase from porcine liver (Sigma) was used as a positive control of hydrolysis.
Proteins were separated using a Novex Tris-Glycine gel (Thermo Fisher Scientific) and Tris-Glycine Native Sample Buffer (Thermo Fisher Scientific). Preparations containing 1.0, 0.1, or 0.05 μg proteins were loaded onto the gel. SeeBlue Pre-stained Protein Standard (Thermo Fisher Scientific) was used as the molecular weight marker. Electrophoresis was conducted at constant 125 V for 3 h at 4°C in 1× Tris-glycine native running buffer (Thermo Fisher Scientific). The gel was then washed twice for 10 min each in 100 mM Tris-HCI, pH 8.0. Esterase activity was detected using the indoxyl acetate assay as described [50 (link)].

Kodama S., Ishizuka J., Miyashita I., Ishii T., Nishiuchi T., Miyoshi H, & Kubo Y. (2017). The morphogenesis-related NDR kinase pathway of Colletotrichum orbiculare is required for translating plant surface signals into infection-related morphogenesis and pathogenesis. PLoS Pathogens, 13(2), e1006189.

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