Tris glycine sds sample buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

Tris-Glycine SDS Sample Buffer is a solution used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to prepare protein samples for analysis. It contains Tris, glycine, and SDS, which denature and solubilize proteins, allowing for their separation based on molecular weight.

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Sample buffer (138 citations) Glycine (132 citations) SDS sample buffer (78 citations) Tris buffer (32 citations) Novex Tris-Glycine SDS Sample Buffer (30 citations) Tris-Glycine-SDS buffer (17 citations) Novex™ Tris-Glycine SDS Sample Buffer (2×) (9 citations) Tris-Glycine SDS Native Sample Buffer (5 citations) Tris-Glycine SDS (2 citations) Tris-Glycine buffer (2 citations)

72 protocols using tris glycine sds sample buffer

Immunoprecipitation of Zonulin Complexes

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To collect the target antigens of CUSABIO and Immundiagnostik zonulin commercial assays, protocols for the immunoprecipitation of the serum antigen-immobilised antibody complex were developed as previously described with modifications [20 (link)]. Undiluted serum samples with high purported zonulin levels, as determined by each respective commercial assay, were selected as antigen sources. Recombinant zonulin was selected as a positive control. Immunoprecipitation buffer, i.e. Novex Tris-Glycine SDS Sample Buffer with 2-mercaptoethanol, was selected as a negative control. Biotinylated zonulin tracer, which competes with serum antigen in binding to immobilised plate antibodies of the Immundiagnostik assay, was also subject to the immunoprecipitation protocol to determine any potential interactions.

Ajamian M., Steer D., Rosella G, & Gibson P.R. (2019). Serum zonulin as a marker of intestinal mucosal barrier function: May not be what it seems. PLoS ONE, 14(1), e0210728.

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Western Blot Analysis of c-Myc Protein

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Serum samples and standard were diluted in 1:50 PBS. We mixed 10 μL diluted serum with 10 μL Tris-glycine SDS sample buffer (2× Novex) and heated at 85°C for 10 min. 20 μL treated sample was run on Novex 12% Tris-glycine gels (Invitrogen, Carlsbad, CA, USA) using Tris-glycine SDS running buffer (Invitrogen, Carlsbad, CA, USA). The protein was then transferred to nitrocellulose membranes using an i-Blot transfer device (Invitrogen, Carlsbad, CA, USA). Membranes were blocked for 1 h at room temperature with Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA) before being probed overnight with primary antibodies (1:1,000 dilution) (goat c-myc antibody; GeneTex catalog no. 30518). Infrared dye (IR)-labeled secondary antibodies (1:5,000 dilution) were then applied using IRDye 680LT Donkey Anti-Goat IgG (H + L). Blots were visualized using the Odyssey Infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA), and images were processed using an image studio program. All antibodies were used at the manufacturer-recommended dilutions.

Gruntman A.M., Gernoux G., Tang Q., Ye G.J., Knop D.R., Wang G., Benson J., Coleman K.E., Keeler A.M., Mueller C., Chicoine L.G., Chulay J.D, & Flotte T.R. (2019). Bridging from Intramuscular to Limb Perfusion Delivery of rAAV: Optimization in a Non-human Primate Study. Molecular Therapy. Methods & Clinical Development, 13, 233-242.

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Monitoring Aβ-Induced TTR Destabilization

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For monitoring Aβ-induced TTR tetramer destabilization, TTR or TTR with hRBP (3.6 μM) was mixed with Aβ40 monomers (60 μM) in PBSA and then incubated for 0–50 h at 37 °C to induce Aβ40 aggregation. Samples were diluted into Tris-glycine SDS sample buffer (Novex) and briefly centrifuged. Control TTR and TTR/hRBP samples were boiled for 10 min to use as RBP and monomeric TTR standards on the gel. Samples were loaded in a 10 to 20% polyacrylamide gradient gel (Novex), electrophoresed in Tris-glycine SDS running buffer (Novex), and stained with Coomassie blue. Destained gels were photocopied, and the densitometry of each protein bands was quantified using ImageJ. The percentage of total TTR tetramer dissociation was determined by the density ratio between the amount of monomeric TTR and total TTR protein. It was reported as the mean and standard deviation of three replicate gel assay experiments.

Mangrolia P, & Murphy R.M. (2018). Retinol-Binding Protein Interferes with Transthyretin-Mediated β-Amyloid Aggregation Inhibition. Biochemistry, 57(33), 5029-5040.

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Whole Cell Lysate and Chromatin Fractionation

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To prepare whole cell lysates, cells were lysed with CellLytic^™M lysis reagent (C2978, Sigma-Aldrich). After thorough mixing and incubation at 4°C for 30 min, lysates were centrifuged at 15,000 g at 4°C for 10 min, and supernatants were collected. To prepare chromatin bound subcellular fraction, we followed the protocol of Subcellular Protein Fractionation Kit from Thermo Scientific (78840). Samples were mixed with tris-glycine SDS sample buffer (Novex, LC2676) and loaded onto Novex tris-glycine gels (Novex). Blotted membrane was blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich. A9418) in phosphate-buffered saline (PBS) with 0.1% tween-20 (PBST). The primary antibodies were diluted in 5% BSA/PBST by 1:3000 for phospo-S345-CHK1, CHK1, phspho-S4/S8-RPA2, RPA1, CtIP, CDC45, DHX9, cyclin A and ORC2, and 1:10000 for phospho-S139-H2AX, SLFN11 (D-2), RPA2, Histone H3, GAPDH, PCNA, MCM3 and MCM2. The secondary antibodies were diluted in 5% non-fat milk by 1:10000. Quantification of band intensity was done using ImageJ software. A proper size of square that was slightly larger than blot bands was set, and used to measure the mean intensity of each band. The square size was consistent through the experiment for each antibody. Intensity of background was subtracted, and the intensity of each control sample was set as 1.

Murai J., Tang S.W., Leo E., Baechler S.A., Redon C.E., Zhang H., Al Abo M., Rajapakse V.N., Nakamura E., Miller Jenkins L.M., Aladjem M.I, & Pommier Y. (2018). SLFN11 blocks stressed replication forks independently of ATR. Molecular cell, 69(3), 371-384.e6.

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Western Blot Analysis of Hemoglobin S-Glutathionylation

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The effectiveness of hemoglobin S-glutathionylation was assessed using Western blot analysis. After the incubation of hemoglobin with GSSG, a Tris-Glycine SDS sample buffer (“Novex”, Carlsbad, CA, USA) was added without mercaptoethanol. Tris-glycine electrophoresis was performed in 14% of PAAG. Before incubation in a blocking buffer (5% milk), the membranes were fixed with a 5% formalin solution for 40 min to reduce the loss of alpha and beta Hb subunits (12–13 kDa). Primary mouse Anti-Glutathione antibody (“Millipore”, MAB5310 1:1000, Temecula, CA, USA) was used for the detection of S-glutathionylated proteins and rabbit anti-human Recombinant Anti-Hemoglobin subunit beta/ba1 antibody (ab92492, Abcam 1:1000, Germany) for the detection of Hb.

Kuleshova I.D., Zaripov P.I., Poluektov Y.M., Anashkina A.A., Kaluzhny D.N., Parshina E.Y., Maksimov G.V., Mitkevich V.A., Makarov A.A, & Petrushanko I.Y. (2023). Changes in Hemoglobin Properties in Complex with Glutathione and after Glutathionylation. International Journal of Molecular Sciences, 24(17), 13557.

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Western Blot Protein Analysis

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Cells and ventricular tissue were lysed in RIPA buffer containing Halt protease and phosphatase inhibitors (Thermo) by scraping or pulverizing, respectively. Protein concentration was determined by BCA Assay (Pierce). Protein samples were added to Tris-Glycine SDS Sample Buffer (Novex) containing Bolt Reducing Buffer, boiled for five minutes, and loaded onto Bis-Tris 4–12% gradient gels (Invitrogen). After separation by electrophoresis, proteins were transferred onto nitrocellulose membranes. Blocking was in 5% milk for one hour at room temperature. Primary antibodies (dilutions and vendors in Supplemental Table 2) were then added to 5% BSA in TBS-T and incubated overnight at 4 °C. Membranes were washed with TBS-T and then incubated with HRP-linked or near infrared dye (NIR) secondary antibodies (Supplemental Table 2) in 5% milk for one hour at room temperature. Membranes were washed with TBS and, for chemiluminescent secondaries, incubated briefly in ECL substrate (Perkin Elmer), and imaged on an ImageQuant LAS 4000 or Azure c600.

Martin T.G., Hunt D.R., Langer S.J., Tan Y., Ebmeier C.C., Crocini C., Chung E, & Leinwand L.A. (2024). A Conserved Mechanism of Cardiac Hypertrophy Regression through FoxO1. bioRxiv.

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Purification and Analysis of Influenza A Virus

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Influenza A virus, A/Puerto Rico/8/34 (H1N1) (PR8), was prepared, as previously reported^{9 (link)}. Purified PR8 virions (~5 mg mL⁻¹) were lysed in 50 mM Tris-HCl buffer (pH 8.0) containing 100 mM KCl, 5 mM MgCl₂, 1 mM dithiothreitol (DTT), 2% Triton X-100, 5% glycerol, 2% lysolecithin and 1 U μL⁻¹ RNasin Plus RNase inhibitor (Promega, Madison, WI, USA) for 1 h at 30 °C. The sample was ultracentrifuged through a 30–70% (w/v) glycerol gradient in Tris-NaCl buffer [50 mM Tris-HCl (pH 8.0) and 150 mM NaCl] at 45,000 rpm for 3 h at 4 °C in a SW55Ti rotor (Beckman Coulter, Brea, CA, USA). Collected fractions were mixed with 2× Tris-glycine SDS sample buffer (Novex; Invitrogen, Carlsbad, CA, USA) and then subjected to SDS-PAGE using a 4–15% Mini Protean TGX precast gel (Bio-Rad Laboratories, Hercules, CA, USA).

Nakano M., Sugita Y., Kodera N., Miyamoto S., Muramoto Y., Wolf M, & Noda T. (2021). Ultrastructure of influenza virus ribonucleoprotein complexes during viral RNA synthesis. Communications Biology, 4, 858.

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Western Blot Analysis of B-Raf and C-Raf

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At 24 h post-infection, cells were collected for western blot analysis. Media was removed, cells washed with PBS, lysed with Blue Lysis Buffer [Mixture of 20 ml T-PER reagent (Thermo Scientific Pierce), 30 ml 2× Tris-glycine SDS sample buffer (Invitrogen), 1.3 ml 1 M DTT, 200 μl 0.5 M EDTA pH 8.0, 80 μl 0.1 M Na₃VO₃, 400 μl 0.1 M NaF and 1 complete protease inhibitor cocktail (1 × Halt cocktail, Pierce] and boiled for 10 min. Western blot analysis were performed as previously described (Austin et al., 2012 (link)). In brief, membranes were incubated with primary antibodies against B-Raf (1:1000; Abcam # ab117860), C-Raf (1:1000; Abcam # ab124452), or actin, diluted in blocking buffer (PBS containing 3% milk and 0.1% Tween-20) overnight, 4°C. Next day, blots were incubated with appropriate secondary antibodies conjugated to HRP, goat anti-rabbit IgG or goat anti-mouse IgG (1:1000; Thermo Scientific; #32460 (rabbit), # 2430 (mouse) in blocking buffer).

Benedict A., Bansal N., Senina S., Hooper I., Lundberg L., de la Fuente C., Narayanan A., Gutting B, & Kehn-Hall K. (2015). Repurposing FDA-approved drugs as therapeutics to treat Rift Valley fever virus infection. Frontiers in Microbiology, 6, 676.

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FFPE Tissue Protein Extraction and Preparation

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PDX FFPE blocks were sectioned at 8 μm onto glass slides, continued with the sectioned deparaffinization in xylene (2–6 section per samples), followed by rehydration in 100% and 95% ethanol. Next, tissue slides were briefly fixed in 70% ethanol containing protease inhibitors (Complete Mini EDTA-free, Roche, Indianapolis, IN, USA), then dehydrated in the series of 95% ethanol, 100% ethanol. Further, sample was lysed directly from slides in an appropriate volume of extraction buffer containing 50% Tissue Protein Extraction Reagent (T-PER, Thermo Fisher Scientific; Waltham, MA, USA), 47.5% 2 × Tris–Glycine SDS sample buffer (Invitrogen), and 2.5% β-mercaptoethanol (Thermo Fisher Scientific; Waltham, MA, USA). The resulting whole tissue lysates were heated for 8 min at 100 °C. Sample lysates was stored at − 80 °C. Prior to lysate printing, samples were reheated at 100 °C for 2 min, vortexed and briefly centrifuged.

Damayanti N.P., Saadatzadeh M.R., Dobrota E., Ordaz J.D., Bailey B.J., Pandya P.H., Bijangi-Vishehsaraei K., Shannon H.E., Alfonso A., Coy K., Trowbridge M., Sinn A.L., Zhang Z.Y., Gallagher R.I., Wulfkuhle J., Petricoin E., Richardson A.M., Marshall M.S., Lion A., Ferguson M.J., Balsara K.E, & Pollok K.E. (2023). Establishment and characterization of patient-derived xenograft of a rare pediatric anaplastic pleomorphic xanthoastrocytoma (PXA) bearing a CDC42SE2-BRAF fusion. Scientific Reports, 13, 9163.

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FFPE-Embedded PDX Tissue RPPA Analysis

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FFPE-embedded xenograft tissue blocks were sectioned at 8 μm onto glass slides. RPPA analysis was conducted on original P0 tumors when available and their respective PDXs for HT72, HT77, HT87, HT96, HT74, HT98, HT120, and HT139. Between 2 and 6 sections per sample were de-paraffinized in xylene, rehydrated in 100% and 95% ethanol, briefly fixed in 70% ethanol containing protease inhibitors (Complete Mini EDTA-free, Roche, Indianapolis, IN, USA), then dehydrated in 95% ethanol, 100% ethanol and then lysed directly from slides in an appropriate volume of extraction buffer containing 50% Tissue Protein Extraction Reagent (T-PER, Thermo Fisher Scientific; Waltham, MA, USA), 47.5% 2× Tris-Glycine SDS sample buffer (Invitrogen, Waltham, MA, USA), and 2.5% β-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA, USA). The resulting whole-tissue lysates were heated for 8 min at 100 °C. Samples were stored at −80 °C and reheated at 100 °C for 2 min, vortexed, and briefly centrifuged just prior to printing.

Pandya P.H., Jannu A.J., Bijangi-Vishehsaraei K., Dobrota E., Bailey B.J., Barghi F., Shannon H.E., Riyahi N., Damayanti N.P., Young C., Malko R., Justice R., Albright E., Sandusky G.E., Wurtz L.D., Collier C.D., Marshall M.S., Gallagher R.I., Wulfkuhle J.D., Petricoin E.F., Coy K., Trowbridge M., Sinn A.L., Renbarger J.L., Ferguson M.J., Huang K., Zhang J., Saadatzadeh M.R, & Pollok K.E. (2022). Integrative Multi-OMICs Identifies Therapeutic Response Biomarkers and Confirms Fidelity of Clinically Annotated, Serially Passaged Patient-Derived Xenografts Established from Primary and Metastatic Pediatric and AYA Solid Tumors. Cancers, 15(1), 259.

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