Tris acetate gel

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

Tris-Acetate gels are a type of electrophoresis gel used for the separation and analysis of proteins and nucleic acids. They provide a matrix for the migration of these biomolecules under an electric field, allowing their separation based on size and charge characteristics.

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135 protocols using tris acetate gel

Kinase-mediated regulation of MEX-5 via PAR-1 and KA1 domains

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Time course kinase reactions were performed with 40 nM MBP::PAR-1 or MBP::PAR-1(ΔKA1) at 30°C in the presence of P³²-ATP for indicated times in a kinase reaction buffer [20 mM HEPES (pH 7.4), 8 µM MgCl₂, 4 µM ATP 1 mg/ml BSA, 15% w/v glycerol] with 1 µM MBP::MEX-5(445-468). All reactions were stopped by the addition of 4× Laemmli SDS buffer. Proteins were resolved using SDS PAGE on two 7% Tris-Acetate gels (Thermo Fisher Scientific). One gel was stained with Coomassie and an image was taken with an iPhone (Apple). Phosphorylation of MEX-5 was visualized using autoradiography of the second unstained gel. KA1 trans-inhibition reactions were performed with 400 nM MBP::PAR-1(ΔKA1) at 30°C for 10 min in a kinase reaction buffer [20 mM HEPES (pH 7.4), 8 µM MgCl₂, 4 µM ATP, 1 mg/ml BSA, 15% w/v glycerol] and 5 µM MBP::MEX-5(445-468). Titration of recombinant KA1 domain protein was carried out with His(6)::KA1 or His(6)::KA1(KRSS) at 0, 5 mM, 20 mM and 50 mM concentrations. Reactions were performed at 30°C in the presence of P³²-ATP for 10 min. All reactions were stopped by the addition of 4× Laemmli SDS buffer. Proteins were resolved using SDS PAGE on a 7% Tris-Acetate gel (Thermo Fisher Scientific). The gel was stained with Coomassie and an image was taken with an iPhone (Apple). Phosphorylation of MEX-5 was visualized by autoradiography of the stained gel.

Folkmann A.W, & Seydoux G. (2019). Spatial regulation of the polarity kinase PAR-1 by parallel inhibitory mechanisms. Development (Cambridge, England), 146(6), dev171116.

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HU-Induced FANCD2 Expression in Mouse ESCs

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Mouse ESCs were harvested after exposure to 1 mM hydroxy urea (HU) for 24 hours. Western blotting was performed using 3–8% Tris-Acetate gels (ThermoFisherScientific). The antibody against mouse FANCD2 was kindly provided by K. J. Patel.
The datasets generated during the current study are available from the corresponding authors on reasonable request.

van de Vrugt H.J., Harmsen T., Riepsaame J., Alexantya G., van Mil S.E., de Vries Y., Bin Ali R., Huijbers I.J., Dorsman J.C., Wolthuis R.M, & te Riele H. (2019). Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair. Scientific Reports, 9, 768.

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Intracellular Collagen Folding and Proteolysis

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Intracellular collagen folding assays were performed as described [51 (link)]. Samples were electrophoresed on 3–8% Tris-acetate gels (ThermoFisher Scientific, Waltham, MA) and quantitated by densitometry of autoradiograms. Assays were performed in duplicate on two independent cultures for each genotype.
To test for proteolytic susceptibility, trypsin digestions (100 μg/ml) of steady-state labeled procollagens redissolved in 100 mM Tris-HCl, pH 7.4 and 400 mM NaCl were performed at 37°C for the indicated times. Digestions were stopped with the addition of trypsin inhibitor (500 μg/ml), and samples were analyzed by SDS-Urea-PAGE.

Cabral W.A., Fratzl-Zelman N., Weis M., Perosky J.E., Alimasa A., Harris R., Kang H., Makareeva E., Barnes A.M., Roschger P., Leikin S., Klaushofer K., Forlino A., Backlund P.S., Eyre D.R., Kozloff K.M, & Marini J.C. (2020). Substitution of murine type I collagen A1 3-hydroxylation site alters matrix structure but does not recapitulate osteogenesis imperfecta bone dysplasia. Matrix biology : journal of the International Society for Matrix Biology, 90, 20-39.

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Quantitative Western Blot Protocol for CFTR

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Cells were lysed with RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate). Total protein amount was determined by the bicinchoninic acid (BCA) assay, following manufacturer's instructions (Thermo Fisher Scientific). Cell lysates were separated on 3%–8% TRIS-Acetate gels (Thermo Fisher Scientific) and transferred to polyvinylidene fluoride (PVDF) blotting membranes (Bio-Rad). Membranes were blocked in Western Breeze blocking buffer (Thermo Fisher Scientific) and probed with antibodies against CFTR (R&D Systems MAB25031; 1:2.000) and Hsp90 (Origene TA500494; 1:15.000). Horseradish peroxidase (HRP)-conjugated anti-mouse antibody (1:10.000; ab6820; Abcam) was used as secondary antibody. Blots of CFTR were developed using Super Signal West Femto (Thermo Fischer Scientific), and of Hsp90 using Luminata Forte Western HRP Substrate (Millipore).

Trepotec Z., Geiger J., Plank C., Aneja M.K, & Rudolph C. (2019). Segmented poly(A) tails significantly reduce recombination of plasmid DNA without affecting mRNA translation efficiency or half-life. RNA, 25(4), 507-518.

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Western Blot Analysis of eIF2α Phosphorylation

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Equal volumes (5 μl) of the in vitro reactions were loaded on 3–8% Tris-Acetate gels (ThermoFisher Scientific). Proteins were transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked in 5% skimmed milk in TBS-T (0.05% Tween) and then probed with following primary antibodies diluted in 4% BSA in TBS-T (0.05% Tween): p-eIF2α (Abcam, #ab32157, 1:1000), eIF2α (Cell Signaling, #L57A5, 1:1000), GCN2 (Cell Signaling, #E9H6C, 1:1000). Next, membranes were washed 3x in TBS-T (0.05% Tween) and incubated with appropriate secondary antibodies: Goat anti-Mouse Alexa Fluor 680 (Invitrogen, #A32729, 1:5000) or Goat anti-Rabbit Alexa Fluor 790 (Invitrogen, #A27041, 1:10,000). Proteins were visualized using the LICOR Odyssey infrared imaging system. The detection of p-eIF2α and eIF2α proteins was performed simultaneously. Bands were quantified using ImageStudioLite and analyses were performed using GraphPad software.

Szaruga M., Janssen D.A., de Miguel C., Hodgson G., Fatalska A., Pitera A.P., Andreeva A, & Bertolotti A. (2023). Activation of the integrated stress response by inhibitors of its kinases. Nature Communications, 14, 5535.

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Immunoblot Detection of Influenza Proteins

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For detection of influenza proteins, samples were denatured and electrophoresed on 3–8% Tris-acetate gels (Thermo Fisher Scientific). Proteins were transferred onto polyvinylidene difluoride membrane (Thermo Fisher Scientific), probed with primary and secondary horseradish peroxidase (HRP)-conjugated antibody and then detected using enzyme chemiluminescent reagents (GE Healthcare). The primary antibody used for immunoblotting was rabbit polyclonal anti-H1N1 influenza A virus nucleocapsid protein (Ab104870, Abcam). The secondary antibody was goat anti-rabbit immunoglobulin HRP (P0448, Dako). Influenza vaccine (Split Virion BP 2015/2016) was used as a positive control.

Menikou S., McArdle A.J., Li M.S., Kaforou M., Langford P.R, & Levin M. (2020). A proteomics-based method for identifying antigens within immune complexes. PLoS ONE, 15(12), e0244157.

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Protein Extraction and Analysis

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Cells were lysed in RIPA buffer for western blot analysis or in RIPA IP buffer for co-immunoprecipitation (Co-IP). Anti-FLAG M2 Magnetic beads (Sigma-Aldrich, MO) eluted FLAG-tagged proteins. Total protein lysates or eluates of the anti-FLAG M2 beads were resolved on NuPAGE 10% Bis-Tris or 3–8% Tris-acetate gels (Thermo Fisher Scientific, MA).

Sun N., Nasello C., Deng L., Wang N., Zhang Y., Xu Z., Song Z., Kwan K., King R.A., Pang Z.P., Xing J., Heiman G.A, & Tischfield J.A. (2017). The PNKD gene is associated with Tourette Disorder or Tic disorder in a multiplex family. Molecular psychiatry, 23(6), 1487-1495.

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Immunoblotting for ISGylation and TAP-nsp123 Cleavage

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Samples assessing ISGylation were lysed in 1% NP40 lysis buffer (100 mM Tris pH 7.5, 100 mM NaCl ,1% NP40) supplemented with 10 mM NEM, 170 μg/mL PMSF, 2 μg/mL leupeptin, 2 μg/mL aprotinin and 10 mM DTT for 10 minutes on ice followed by a 10 minute spin at 20,000 x g. Samples were run on BOLT 4-12% Bis-Tris gels (Thermo) and blotted with anti-M2 FLAG antibody (Sigma) and anti-actin (Invitrogen AC-15). Samples assessing the effect of 6-TG on TAP-nsp123 cleavage were lysed in RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 0.5% NP40, 0.1% SDS, 0.5% Sodium deoxycholate w/v) supplemented with 10 mM NEM, 170 μg/mL PMSF, 2 μg/mL leupeptin, 2 μg/mL aprotinin, 10 mM DTT, and 2 units/mL Universal Nuclease (Pierce) and incubated for 10 minutes on ice followed by a 10 minute spin at 20,000 x g. Samples were run on 3-8% Tris-Acetate gels (Thermo) and blotted for TAP using anti-protein A antibody (Sigma) and anti-actin (Invitrogen AC-15).

Swaim C.D., Dwivedi V., Perng Y.C., Zhao X., Canadeo L.A., Harastani H.H., Darling T.L., Boon A.C., Lenschow D.J., Kulkarni V, & Huibregtse J.M. (2021). 6-Thioguanine blocks SARS-CoV-2 replication by inhibition of PLpro. iScience, 24(10), 103213.

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Western Blot Protein Analysis Protocol

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Tissues were isolated quickly, frozen in liquid nitrogen, and stored at −80 °C until use. For western blotting studies, frozen tissues were homogenized in ice-cold RIPA buffer (Sigma Aldrich) and protein concentrations were determined using a BCA protein assay (Thermo Fisher Scientific). Protein extracts were separated on NuPAGE 4–12% Bis-Tris or, for high molecular mass protein targets, on 3–8% Tris-Acetate gels (Thermo Fisher Scientific) and blotted onto nitrocellulose membranes (Bio-Rad). Membranes were blocked for 1 h at room temperature in TBS-T (0.1%) containing 5% BSA. Membranes were then incubated overnight with primary antibodies at 4 °C. Following three washing steps in TBS-T (0.1%), membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. After thorough washing, proteins were visualized with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) on the c600 Imaging System (Azure Biosystems). Immunoreactive bands were quantified using Image J Software (NIH). Uncropped images are provided in the Source Data file.

Meister J., Bone D.B., Knudsen J.R., Barella L.F., Velenosi T.J., Akhmedov D., Lee R.J., Cohen A.H., Gavrilova O., Cui Y., Karsenty G., Chen M., Weinstein L.S., Kleinert M., Berdeaux R., Jensen T.E., Richter E.A, & Wess J. (2022). Clenbuterol exerts antidiabetic activity through metabolic reprogramming of skeletal muscle cells. Nature Communications, 13, 22.

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Quantitative Western Blotting of Dystrophin and Utrophin

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Samples for dystrophin and utrophin westerns were prepared, and protein levels quantified as previously described (43 (link)). Briefly, homogenised samples were run on 3–8% Tris-acetate gels (Thermo Fisher Scientific), blotted onto PVDF membrane (Millipore), and probed with DYS1 (Novocastra, dystrophin) or MANCHO antibody (KED laboratory—Oxford, utrophin). Blots were visualised using IRDye 800CW goat-anti mouse IgG (LI-COR) on the Odyssey imaging system. For dystrophin, vinculin (hVIN-1; Sigma-Aldrich) was used as loading control, and for utrophin, the samples were quantified against total protein using Coomassie stain (Sigma-Aldrich).

Betts C.A., Jagannath A., van Westering T.L., Bowerman M., Banerjee S., Meng J., Falzarano M.S., Cravo L., McClorey G., Meijboom K.E., Bhomra A., Lim W.F., Rinaldi C., Counsell J.R., Chwalenia K., O’Donovan E., Saleh A.F., Gait M.J., Morgan J.E., Ferlini A., Foster R.G, & Wood M.J. (2021). Dystrophin involvement in peripheral circadian SRF signalling. Life Science Alliance, 4(10), e202101014.

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