Nupage 3 8 tris acetate gel

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

NuPAGE 3–8% Tris-Acetate gels are precast polyacrylamide gels used for electrophoretic separation of proteins. They have a Tris-acetate buffer system and are designed for the separation of high molecular weight proteins.

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NuPAGE 3–8% Tris-Acetate (FANCD2) polyacrylamide gels NuPAGE Tris-acetate 3–8 % gels NUPAGE Novex 3-8% Tris-acetate gradient gels NuPAGE 3–8% Tris-acetate pre-cast gels NuPAGE 3-8% Tris-Acetate Tris-Acetate gels NuPAGE gels NuPAGE

74 protocols using nupage 3 8 tris acetate gel

Protein Extraction and Western Blot Analysis

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Cells were lysed in RIPA buffer with protease/phosphatase inhibitor
(cat# 5872, Cell Signaling Technology, Danvers, MA) or Laemmli Sample Buffer
(cat# 1610747, Biorad, Hercules, CA) by sonication. PAGE was carried out
with manufacturer-recommended buffers on 4-20% or 7.5% Mini TGX Gels
(Biorad), NuPAGE 4-12% BisTris gels, or NuPAGE 3-8% Tris-Acetate gels
(Thermo). Semi-dry transfers were carried out for Biorad gels or NuPAGE
BisTris gels at 2.5A for 5-15 minutes using the Trans-Blot-Turbo (Biorad).
Wet transfers were carried out for Tris-Acetate gels at 30V overnight. All
blocking was performed with Odyssey Blocking Buffer (Licor). Primary
antibodies were incubated with membranes overnight at 4C, then
infrared-conjugated secondaries (Licor) were added 1:10,000 and imaged on a
Licor Odyssey Scanner. Quantifications were carried out using Image Studio
v4.0. Blots were stripped with Reblot Plus Strong Solution (Millipore
Sigma). A list of antibodies used can be found in the Supplementary Methods Key Reagents
table.

Ardeljan D., Steranka J.P., Liu C., Li Z., Taylor M.S., Payer L.M., Gorbounov M., Sarnecki J.S., Deshpande V., Hruban R.H., Boeke J.D., Fenyö D., Wu P.H., Smogorzewska A., Holland A.J, & Burns K.H. (2020). Cell fitness screens reveal a conflict between LINE-1 retrotransposition and DNA replication. Nature structural & molecular biology, 27(2), 168-178.

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Protein Extraction and Western Blot Analysis

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Native PAGE analysis of BMDMs

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BMDMs were stimulated as described and lysed in RIPA lysis buffer without SDS. Lysates were centrifuged at 2,300 ×g for 10 min to pellet DNA. Supernatant was centrifuged at 16,100 ×g for 25 min, and the pellet was resuspended in native PAGE sample buffer (Thermo Fisher Scientific). The samples were loaded onto NuPage 3%–8% Tris-acetate gels (Thermo Fisher Scientific) without boiling, and native PAGE was conducted using Tris-glycine running buffer (Thermo Fisher Scientific). The gel was soaked in 10% SDS solution for 10 min before performing semidry transfer and continuing with conventional immunoblot.

Bittner Z.A., Liu X., Mateo Tortola M., Tapia-Abellán A., Shankar S., Andreeva L., Mangan M., Spalinger M., Kalbacher H., Düwell P., Lovotti M., Bosch K., Dickhöfer S., Marcu A., Stevanović S., Herster F., Cardona Gloria Y., Chang T.H., Bork F., Greve C.L., Löffler M.W., Wolz O.O., Schilling N.A., Kümmerle-Deschner J.B., Wagner S., Delor A., Grimbacher B., Hantschel O., Scharl M., Wu H., Latz E, & Weber A.N. (2021). BTK operates a phospho-tyrosine switch to regulate NLRP3 inflammasome activity. The Journal of Experimental Medicine, 218(11), e20201656.

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Protein Complex Immunoprecipitation in S2 Cells

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S2 cells were transfected with pMT-N-EGFP, pMT-Flag-Ubi, and pMT-Dx-V5 (WT or ΔRF). After 48 h, 1 mM CuSO₄ was added to induce protein expression, followed by incubation at 18°C for 24 h. The cells were lysed by lysis buffer (50 mM Tris-HCl, pH7.5, 125 mM NaCl, 5% glycerol, 0.5% NP-40, 1.5 mM MgCl2, 1 mM DTT, 1 mM EGTA, 1 mM N-ethyl-maleimide, 10 μM MG132, and Halt Protease and Phosphatase Inhibitor Cocktail [Thermo Fisher Scientific]), and pulled-down with GFP-Trap (ChromoTek). The immunoprecipitated samples and 10% lysates were separated on NuPage 3–8% Tris-Acetate gels (Thermo Fisher Scientific) and transferred to PVDF membranes (Merck). Primary antibodies used for Western blotting were rabbit anti-GFP (50430-2-AP, 1:5,000; Proteintech), mouse anti-Flag (M2, 1:5,000; Merck), mouse anti-V5 (OASA04489, 1:5,000; Aviva System Biology), and mouse anti-Peanut ((Cat# 4C9H4 anti-peanut, RRID:AB_528429, 1:10,000; DSHB), and staining was detected by LI-COR Odyssey imaging system with anti-mouse Alexa Fluor Plus 800 (Cat# A32730, RRID:AB_2633279; Thermo Fisher Scientific) and anti-rabbit Alexa Fluor Plus 680 (Cat# A32734, RRID:AB_2633283; Thermo Fisher Scientific) secondary antibodies, used at 1:10,000.

Shimizu H., Hosseini-Alghaderi S., Woodcock S.A, & Baron M. (2024). Alternative mechanisms of Notch activation by partitioning into distinct endosomal domains. The Journal of Cell Biology, 223(5), e202211041.

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Immunoblot Analysis of Protein Extracts

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Total protein extracts for immunoblot analysis were prepared in Cell lysis buffer (Cell Signaling Technology) with freshly added protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland) by homogenizing cells. Next, an equal amount of protein lysates was separated on 4–12% Bis-Tris gels (Thermo Fisher Scientific, Waltham, MA, USA) or NuPAGE 3–8% Tris-Acetate gels (Thermo Fisher Scientific) and was transferred to a polyvinylidene difluoride (PVDF, Thermo Fisher Scientific) membrane. The membranes were blocked with TBS blocking buffer containing 5% skim milk (BD Biosciences) or 2% bovine serum albumin (BSA) for 1 h and incubated with primary antibodies for 12 h at 4 °C. Detection was carried out using an ECL-Plus kit (Amersham, Buckinghamshire, UK). Samples from three independent experiments were used, and the relative expression levels were determined using a Fusion-FX instrument (Viber Lourmat, Collegien, France).

Jeon Y.M., Kwon Y., Lee S., Kim S., Jo M., Lee S., Kim S.R., Kim K, & Kim H.J. (2021). Vitamin B12 Reduces TDP-43 Toxicity by Alleviating Oxidative Stress and Mitochondrial Dysfunction. Antioxidants, 11(1), 82.

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Evaluating Degrader Compound Effects on HEK293 Cells

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HEK293 cells were obtained from Genentech’s repository and were cultured under standard conditions in RPMI medium containing 10% FBS. For evaluating the cellular effects of the degrader compounds, 0.4 million cells were seeded in 1 well of a 12-well plate. The following day, the medium was diluted twofold with fresh medium containing vehicle (DMSO) or compounds. For degradation rescue studies, cells were co-treated with the proteasome inhibitor bortezomib (62.5 nM) or BET ligand (0.1, 1 or 10 µM). Cells were cultured with the compounds for an additional 20 h and collected. Complete cell lysates were prepared in urea lysis buffer (50 mM Tris-HCl (pH 7.4), 120 mM NaCl, 1 mM EDTA, 1 % NP-40, 6 M urea and 1× Roche cOmplete protease inhibitor cocktail). Ten micrograms of total lysate was resolved by NuPAGE 3–8% Tris-acetate gels (Thermo Fisher) and transferred to nitrocellulose membranes (Bio-Rad Trans-Blot transfer system) followed by western blotting analysis. Primary antibodies were diluted 1:3,000 and incubated for 1 h at room temperature or 4 °C overnight. Secondary antibodies were diluted 1:10,000 and incubated for 60 min at room temperature. Blots were imaged by scanning using LICOR Odyssey CLx.

Bashore C., Prakash S., Johnson M.C., Conrad R.J., Kekessie I.A., Scales S.J., Ishisoko N., Kleinheinz T., Liu P.S., Popovych N., Wecksler A.T., Zhou L., Tam C., Zilberleyb I., Srinivasan R., Blake R.A., Song A., Staben S.T., Zhang Y., Arnott D., Fairbrother W.J., Foster S.A., Wertz I.E., Ciferri C, & Dueber E.C. (2022). Targeted degradation via direct 26S proteasome recruitment. Nature Chemical Biology, 19(1), 55-63.

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Detecting Sumoylated Proteins using Ni-NTA

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Sumoylated proteins were pulled down from cells containing 8His-tagged SUMO expressed from its endogenous locus using the standard Ni-NTA pull-down method as previously described (Ulrich & Davies, 2009 (link)). Briefly, protein extracts prepared in 55% TCA were incubated in buffer A (6 M guanidine HCl, 100 mM sodium phosphate at pH 8.0, 10 mM Tris-HCl at pH 8.0) with rotation for 1 h at room temperature. The cleared supernatant was obtained after centrifuging for 20 min and was then incubated overnight at room temperature with Ni-NTA resin (Qiagen 30210) in the presence of 0.05% Tween-20 and 4.4 mM imidazole with rotation. Beads were washed twice with buffer A supplemented with 0.05% Tween 20 and then four times with buffer C (8 M urea, 100 nM sodium phosphate at pH 6.3, 10 mM Tris-HCl at pH 6.3) supplemented with 0.05% Tween 20. Proteins were eluted from the beads using HU buffer (8 M urea, 200 mM Tris-HCl at pH 6.8, 1 mM EDTA, 5% SDS, 0.1% bromophenol blue, 1.5% DTT, 200 mM imidazole). Samples were loaded onto NuPAGE^™ 3–8% Tris-acetate gels (Thermo Fisher EA03752) for immunoblotting to detect both sumoylated and unmodified Srs2. Equal loading was verified using Ponceau S staining.

Fan J., Dhingra N., Yang T., Yang V, & Zhao X. (2024). Srs2 binding to PCNA and its sumoylation contribute to RPA antagonism during the DNA damage response. bioRxiv.

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Detecting V. vulnificus Toxin RtxA1 in HeLa Cells

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HeLa cells (Korea Cell Line Bank, Seoul, Korea) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Welgene, Daegu, Korea) containing 10% fetal bovine serum (ThermoFisher Scientific, Waltham, MA, US). V. vulnificus strains from cultures grown overnight in LB broth were diluted 200-fold with 10 mL of fresh LB broth in a shaking incubator at 37°C for another 3 h. HeLa cells (5×10⁵ cells/well) grown overnight in 6-well plates were washed with serum free DMEM medium before being infected with bacteria at an MOI of 20 for 120, 150, or 180 min. The supernatants (300 µL) were precipitated by the addition of 3-fold ice-cold acetone. Bacterial pellets of the supernatants and HeLa cells were lysed with the cell lysis buffer (Promega, Madison, WI, USA) containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) shaking on ice for 30 min and harvested by centrifugation at 13000 rpm for 10 min after scrapping. The protein concentration was quantified using the Bradford’s reagent (Bio-Rad Laboratories, USA). Equal amounts of protein were separated by NuPAGE™ 3%-8% Tris-Acetate gels (Thermo Fisher Scientific, Carlsbad, CA, USA) and subsequently subjected to Western blotting using an anti-RtxA1 antibody specific to amino acid 1492-1970 (RTX-D2 domain). Western blotting was performed as described above.

Gong Y., Guo R.H., Rhee J.H, & Kim Y.R. (2021). TolCV1 Has Multifaceted Roles During Vibrio vulnificus Infection. Frontiers in Cellular and Infection Microbiology, 11, 673222.

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Western Blot Analysis of Ataxin-7

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First 25 μg of total protein isolated with PB buffer (60 mM Tris-base, 2% SDS, 10% sucrose, 2 mM PMSF) was run on NuPAGE 3%–8% Tris-Acetate gels (Thermo Fisher Scientific) in Tris-Acetate SDS Running Buffer (Life Technologies) at 4 °C. The immunoreaction was performed using the following antibodies: anti-ataxin-7 (NBP1-42657, Novus Biologicals, Littleton, CO, USA), anti-vinculin (4650, Cell Signaling Technology, Danvers, MA, USA), and anti-rabbit horseradish peroxidase (HRP)-conjugate (Jackson ImmunoResearch, West Grove, PA, USA,) and 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 the Gel-Pro Analyzer (Media Cybernetics, Rockville, MD, USA).

Fiszer A., Wroblewska J.P., Nowak B.M, & Krzyzosiak W.J. (2016). Mutant CAG Repeats Effectively Targeted by RNA Interference in SCA7 Cells. Genes, 7(12), 132.

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Western Blot Analysis of MLL Subunits

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Nuclear and cytoplasmic extracts were prepared using the ThermoScientific NER kit as per manufacturer's instructions. Whole cell extracts were prepared using RIPA buffer. Western blots of MLL^N and MLL^C subunits were carried out on NuPAGE 3–8% Tris-Acetate gels (ThermoScientific) as per the manufacturer's instructions.
Primary antibodies used in this study were as follows: H3 (06-755) and γH2AX (JBW301) (Merck Millipore); Actin (#3700), pan-β-catenin (#8480), Tubulin (#2128), Pin1 (#3722), phospho-AKT Ser473 (#4060) and AKT (#4691) (CST); p27 (610241) (BD Biosciences, Oxford, UK); MLL1 (A300-086A), AF9 (A300-596A), AF6 (A302-199A) and ENL (A302-268A) (Bethyl, UK); MLL^C (sc-374,392), pan-Cks (sc-6238), CDK2 (sc-163), Cleaved-Caspase 3 (sc-7272) and normal IgG (Santa Cruz, CA, USA); and Cks1 (37-0200) and Cks2 (37-0300) (ThermoScientific).

Grey W., Ivey A., Milne T.A., Haferlach T., Grimwade D., Uhlmann F., Voisset E, & Yu V. (2018). The Cks1/Cks2 axis fine-tunes Mll1 expression and is crucial for MLL-rearranged leukaemia cell viability. Biochimica et Biophysica Acta, 1865(1), 105-116.

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