Xt tricine buffer

Manufactured by Bio-Rad

Sourced in United States

The XT Tricine buffer is a laboratory buffer solution designed for the electrophoretic separation of proteins. It is primarily used in the XT Criterion system for the resolution of low-molecular-weight proteins. The buffer provides a stable pH environment and helps maintain the structural integrity of proteins during the separation process.

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Lab products found in correlation

Gel pro analyzer, by Media Cybernetics (2 mentions) Nitrocellulose membrane, by Merck Group (2 mentions) Westernbright quantum hrp substrate, by Advansta (2 mentions) Horseradish peroxidase hrp conjugated goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Nitrocellulose blotting membrane, by GE Healthcare (1 mentions) Westar antares chemiluminescent substrate, by Cyanagen (1 mentions) Anti mouse hrp conjugate, by Merck Group (1 mentions) Snap i d system, by Merck Group (1 mentions) Ab83497, by Abcam (1 mentions) Anti rabbit hrp conjugate, by Jackson ImmunoResearch (1 mentions) Mab2166, by Merck Group (1 mentions) Anti dicer antibody, by Cell Signaling Technology (1 mentions) Dynabeads, by Bio-Rad (1 mentions) Dynabeads protein g, by Thermo Fisher Scientific (1 mentions) Nupage tris acetate sds running buffer, by Thermo Fisher Scientific (1 mentions) Phospho mtor ser2448, by Cell Signaling Technology (1 mentions) Odyssey infrared imaging system, by LI COR (1 mentions) Aprotinin, by Merck Group (1 mentions) Leupeptin, by Merck Group (1 mentions)

7 protocols using xt tricine buffer

Quantification of CCHFV Viral Proteins

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For detection of CCHFV L polymerase (448kDa), lysate samples from tc-VLP donor cells were run through Criterion XT Tris-acetate precast gels (3–8% gradient) (Biorad) in a XT tricine buffer (Biorad), for 1 h 40 min at 150V. Proteins were transferred on an EtOH-activated polyvinylidene fluoride (PVDF) membrane (Millipore) by wet blotting over-night at 40 mA and 4°C, using Tris-glycine transfer buffer (5.8 g/L Tris (Acros), 2 g/L glycine (Roth), 10% absolute EtOH (Roth)). The CCHFV L was detected via its V5-tag (1:5000) (Novex, Life-technologies) and a horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Thermo Fisher) (1:20,000). Quantification of the signals was performed using the Image Lab 4.0 software.
For detection of CCHFV Gn and N in donor cell supernatants, tc-VLPs (and CCHFV strain IbAr10200 as control) were produced, concentrated by ultracentrifugation through a 20% sucrose cushion, and their protein content analyzed by Western blotting (10% SDS-PAGE) and chemiluminescence quantification as described in [32 (link)].
All quantification values are shown as a percentage of the signal in wt L donor cells.

Devignot S., Kromer T., Mirazimi A, & Weber F. (2020). ISG15 overexpression compensates the defect of Crimean-Congo hemorrhagic fever virus polymerase bearing a protease-inactive ovarian tumor domain. PLoS Neglected Tropical Diseases, 14(9), e0008610.

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Protein Extraction and Western Blot Analysis

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Protein was isolated from the cells with the use of PB (60 mM Tris-base, 2% SDS, 10% sucrose, 2 mM PMSF). A total of 30 μg of protein was resolved on a Tris-acetate sodium dodecyl sulfate (SDS)-polyacrylamide gel (1.5 cm, 4% stacking gel/4.5 cm, 5% resolving gel, acrylamide:bis-acrylamide ratio of 49:1) in XT Tricine buffer (Bio-Rad) at 135 V in an ice-water bath. After electrophoresis, the proteins were wet-transferred overnight to a nitrocellulose blotting membrane (GE Healthcare Life Sciences, Chicago, Illinois, IL, USA). The primary antibodies, including anti-huntingtin, anti-plectin, and the anti-rabbit HRP conjugate secondary antibody were used in a PBS/0.1% Tween-20 buffer containing 5% nonfat milk. The immunoreaction was detected using the Westar Antares Chemiluminescent substrate (Cyanagen, Bologna, Italy). The protein bands were scanned directly from the membrane using a camera and quantified using a Gel-Pro Analyzer (Media Cybernetics, Rockville, MD, USA). A list of all antibodies used is provided in Table S7.

Dabrowska M., Ciolak A., Kozlowska E., Fiszer A, & Olejniczak M. (2020). Generation of New Isogenic Models of Huntington’s Disease Using CRISPR-Cas9 Technology. International Journal of Molecular Sciences, 21(5), 1854.

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Western Blot Analysis of Huntingtin Protein

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The western blot analysis for the HTT protein (17/68Q tract) was performed as previously described (Fiszer et al., 2011 (link)). Briefly, 30 μg of total protein was run on a Tris-acetate sodium dodecyl sulfate (SDS)-polyacrylamide gel (1.5 cm, 4% stacking gel/4.5 cm, 5% resolving gel, acrylamide:bis-acrylamide ratio of 49:1) in XT Tricine buffer (Bio-Rad, Hercules, CA, USA) at 130 V in an ice-water bath. Subsequently, the proteins were wet-transferred to a nitrocellulose membrane (Sigma–Aldrich). All of the immunodetection steps were performed using the SNAPid system (Millipore). The primary antibodies anti-huntingtin (1:1000, MAB2166, Millipore) and anti-plectin (1:1000, ab83497, Abcam, Cambridge, UK) and secondary antibodies anti-mouse HRP-conjugate (1:2000, A9917, Sigma–Aldrich) and anti-rabbit HRP-conjugate (1:2000, 711-035-152, Jackson ImmunoResearch) were used in a PBS/0.1% Tween-20 buffer containing 0.25% non-fat milk. The immunoreaction was detected using WesternBright Quantum HRP Substrate (Advansta, Menlo Park, CA, USA). The protein bands were scanned directly from the membrane using a camera and were quantified using Gel-Pro Analyzer.

Urbanek M.O., Fiszer A, & Krzyzosiak W.J. (2017). Reduction of Huntington’s Disease RNA Foci by CAG Repeat-Targeting Reagents. Frontiers in Cellular Neuroscience, 11, 82.

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Dicer-TRBP Complex Isolation and Cleavage Assay

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Prior to Dicer–TRBP complex formation, Dynabeads Protein G (Life Technologies) was used to bind 2 μg anti-TRBP antibody (Santa Cruz sc-514124) for 4 h at room temperature in 0.02% phosphate buffered saline (PBS)-Tween. After washing, 250 μl of reconstituted Dicer–TRBP complex was added to the antibody-conjugated Dynabeads and rotated overnight at 4°C.
Prior to western blotting for Dicer detection, the beads, after washing, were resuspended in a 10 μl of PBS and 10 μl 3× sodium dodecylsulphate (SDS) sample buffer, heated for 5 min at 95°C and loaded onto a 5% Tris-acetate SDS-page (acrylamide:bis = 49:1) in XT Tricine buffer (Bio-Rad) as previously described (41 (link),42 (link)). The blots were probed with anti-Dicer antibody (Cell Signaling Technology, 1:1000) and then HRP-conjugated secondary antibody, anti-rabbit (Sigma, 1:1000). The immunoreactions were detected using Westernbright Quantum HRP substrate (Avansta).
For Dicer–TRBP cleavage assay, the pull-down was performed in the same manner as for Dicer detection, except the beads-Ab-TRBP/Dicer complex, after washing, was resuspended in a 20 μl of PBS.

Starega-Roslan J., Galka-Marciniak P, & Krzyzosiak W.J. (2015). Nucleotide sequence of miRNA precursor contributes to cleavage site selection by Dicer. Nucleic Acids Research, 43(22), 10939-10951.

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Western Blot Analysis of HTT Protein

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Western blot analysis for HTT protein expression isolated from cell culture was performed as previously described.²⁴ (link) Briefly, 30 μg of total protein was separated on a Tris-acetate SDS-polyacrylamide gel (1.5 cm, 4% stacking gel/4.5 cm, 5% resolving gel, acrylamide:bis-acrylamide ratio of 49:1) in XT Tricine buffer (Bio-Rad) at 135 V in an ice-water bath. For proteins isolated from mouse brains, NuPAGE Tris-Acetate 3%–8% Protein Gel (Thermo Fisher Scientific) in NuPAGE Tris-Acetate SDS Running Buffer (Thermo Fisher Scientific) were used. After electrophoresis, the proteins were transferred overnight to a nitrocellulose membrane (Sigma-Aldrich) by the wet transfer method. The primary and secondary antibodies were used in PBS/0.1% Tween 20 buffer containing 5% nonfat milk. Immunoreactions were detected using Western Bright Quantum HRP Substrate (Advansta, Menlo Park, CA). Protein bands were scanned directly from the membrane using a camera, and band densities were quantified using a Gel-Pro Analyzer (Media Cybernetics). Plectin or calnexin was used as the reference protein. A list of all antibodies used is provided in Table S4.

Kotowska-Zimmer A., Przybyl L., Pewinska M., Suszynska-Zajczyk J., Wronka D., Figiel M, & Olejniczak M. (2022). A CAG repeat-targeting artificial miRNA lowers the mutant huntingtin level in the YAC128 model of Huntington's disease. Molecular Therapy. Nucleic Acids, 28, 702-715.

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Analyzing Viral Capsid Composition

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Virus capsid composition was analyzed by standard immunoblotting techniques. Briefly, 60 μg of protein from purified virus stocks were denatured by heating at 95 °C for 5 minutes, fractionated by SDS-PAGE (7% Tris-Acetate gel, XT Tricine buffer, Bio-Rad, Hercules, CA), transferred to a nitrocellulose membrane, blocked with 5% non-fat dry milk, and blotted for 8 hours at 4 °C with anti-AAV Ab clone B1 (mouse, 1:500, American Research Products, Inc., Belmont, MA). Membrane was washed with blocking buffer and incubated with anti-Mouse Ab (1:10000, SCBT, Dallas, TX) conjugated with horseradish peroxidase. Bands were detected with ECL Supersignal (Pierce, Thermo Scientific, Rockford, IL).

Finet J.E., Wan X, & Donahue J.K. (2017). Fusion of Anthopleurin-B to AAV2 increases specificity of cardiac gene transfer. Virology, 513, 43-51.

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mTORC1 Phosphorylation Analysis by Western Blot

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mTORC1 activity is reflected by mTORC1 phosphorylation which was determined by western blot as previously described [38 (link)]. In short, the treated cells were first lysed with radioimmunoprecipitation assay (RIPA) buffer consisting of 20 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet, 1 mM DTT, 1 mM Na vanadate, 1 mM PMSF, 10 µg/mL leupeptin, 1% aprotinin (Sigma-Aldrich). Proteins were separated by electrophoresis using precast Tris acetate gels and XT-tricine buffer (Bio-Rad, Hercules, CA, USA), run at 150 V. The proteins were transferred to nitrocellulose membranes (Bio-Rad) by electroblotting at 100 V for 1 h using the Trans-Blot Turbo system (Bio-Rad). Equal protein loading was checked with PonceauS staining after which the membranes were blocked and overnight probed with the primary antibodies phospho-mTOR-Ser2448 and mTOR from Cell Signaling (Beverly, MA) at 4 °C. Following the secondary antibody and successive washing, protein quantification was performed by scanning using the Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE).

Tayyeb J.Z., Popeijus H.E., van de Sanden J., Zwaan W., Mensink R.P, & Plat J. (2022). Effects of Individual Amino Acids on PPARα Transactivation, mTORC1 Activation, ApoA-I Transcription and pro-ApoA-I Secretion. International Journal of Molecular Sciences, 23(11), 6071.

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