Bis tris gradient gel

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Germany

The 4–12% Bis-Tris gradient gel is a laboratory equipment product designed for protein electrophoresis. It features a gradient of 4% to 12% bis-tris polyacrylamide, which allows for the separation and analysis of a wide range of protein sizes.

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Lab products found in correlation

Pvdf membrane, by Merck Group (7 mentions) Nitrocellulose membrane, by Thermo Fisher Scientific (7 mentions) Dc protein assay, by Bio-Rad (6 mentions) Nitrocellulose membrane, by Bio-Rad (6 mentions) β actin, by Merck Group (6 mentions) Gapdh, by Cell Signaling Technology (5 mentions) Protease inhibitor cocktail, by Roche (5 mentions) Nupage lds sample buffer, by Thermo Fisher Scientific (5 mentions) Ripa buffer, by Thermo Fisher Scientific (5 mentions) Las4000 imagequant, by GE Healthcare (5 mentions) Pierce ecl western blotting substrate, by Thermo Fisher Scientific (5 mentions) Supersignal west pico, by Thermo Fisher Scientific (4 mentions) Complete protease inhibitor cocktail, by Roche (4 mentions) Lds sample buffer, by Thermo Fisher Scientific (4 mentions) Odyssey blocking buffer, by LI COR (4 mentions) Trans blot turbo transfer system, by Bio-Rad (4 mentions) Biomax film, by Kodak (4 mentions) Versadoc imaging system, by Bio-Rad (4 mentions) Protran, by Cytiva (3 mentions) Cell lysis buffer, by Cell Signaling Technology (3 mentions)

Spelling variants of this product

Bis-Tris gels (1248 citations) 4–12% gradient gels (121 citations) NuPAGE-Novex Bis-Tris 4%–12% gradient gels (14 citations) Bolt 4–12% Bis-Tris gradient gels (6 citations) 4–12% bis-Tris gradient mini gels (3 citations) NuPAGE 4–12% (w/v) Bis-Tris gradient gels (2 citations) Gradient 4–12% Bis-Tris pre-cast gels (2 citations) Novex Bis-Tris Midi gradient gels (2 citations) Native PAGE bis-tris gels of gradient 4 to 16%T (2 citations) 4-12% NuPAGE Bis-Tris gradient mini gels (2 citations)

167 protocols using bis tris gradient gel

Western Blot Analysis of Doxycycline-Treated Cells

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Lysates were made from cells treated with or without doxycycline for 3 d by scraping in SDS lysis buffer (4% SDS and 100 mM Tris, pH 6.8). Lysates were quantified, and 30 µg protein was loaded on 4–12% Bis-Tris gradient gels (Invitrogen). Wet transfers were performed onto nitrocellulose paper (Protran NC; GE Healthcare Biosciences), blocked with 5% milk, and incubated with the previously described antibodies for 1 h. Blots were washed 3× for 5 min using TBST (0.25% Tween 20, 20 mM Tris, pH 8.0, and 137 mM NaCl) and incubated with HRP secondary antibody for 1 h. Blots were then washed 3× for 5 min and developed using SuperSignal West Pico or Femto (PI34078, PI34095; Thermo Fisher Scientific) reagent and exposure to film.

Toyama B.H., Arrojo e Drigo R., Lev-Ram V., Ramachandra R., Deerinck T.J., Lechene C., Ellisman M.H, & Hetzer M.W. (2019). Visualization of long-lived proteins reveals age mosaicism within nuclei of postmitotic cells. The Journal of Cell Biology, 218(2), 433-444.

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Regulation of Klotho and FGF23 Signaling

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HEK-293 T cells were treated with or without sKL(3 μM) and FGF23 (1 μM) alone and in combination for 2 h in presence of heparin (10 μg/ml). Then the cells were lysed with 150 μl of M-PER Cell Protein Extraction reagent (Pierce Biotechnology, Rockford, IL, USA) with 1 × Halt protease inhibitor and 1 mM phenylmethanesulfonyl fluoride (PMSF) per well. After three of 30-s sonication, total cell lysates were centrifuged at 13,000 xg for 10 min and supernatants were stored at −80 °C until use. Protein concentrations of the supernatant were determined with a total protein assay kit (Bio-Rad, Hercules, CA). Equal quantities of protein were subjected to 4–12% Bis-Tris gradient Gels (Invitrogen, Carlsbad, CA) and were analyzed with standard western blot protocols (HRP-conjugated secondary antibodies from Santa Cruz Biotechnology and ECL chemiluminescent immunodetection system from GE Healthcare Bio-Sciences). Anti-Phospho-ERK1/2 (Thr202/Tyr204) (D13.14.4E, #4370), anti-ERK1/2 (#9102), anti-Phospho-PLCγ1 (D25A9, #8713), and anti-PLCγ1 (#2822) were purchased from Cell Signaling Technologies (Danvers, MA). Anti-Klotho (KAL-KO604) was purchased from Cosmo Bio USA, Inc. (Carlsbad, CA). Anti-β-actin (sc-47778) antibody was obtained from Santa Cruz Biotechnology (Paso Robles, CA). The intensity of bands was quantified using Image J software (http://rsb.info.nih.gov/ij/).

Han X., Cai C., Xiao Z, & Quarles L.D. (2019). FGF23 induced left ventricular hypertrophy mediated by FGFR4 signaling in the myocardium is attenuated by soluble Klotho in mice. Journal of molecular and cellular cardiology, 138, 66-74.

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Quantitative Immunoblotting of Autophagy-Related Proteins

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Cell lysates were collected in 8M urea/0.2% SDS. A BCA assay (Pierce) was used to determine protein concentration. 5 μg of protein was separated on 4–12% bis-tris gradient gels (Invitrogen) and transferred onto nitrocellulose membrane. Revert Total Protein Stain (LI-COR Biosciences) was used to assess protein loading. The following primary antibodies/dilutions were used: BAG3 (Proteintech 10599–1AP, 1:4000), HSP70 (Proteintech 10995–1AP, 1:4000), HSPB8 (Proteintech 15287–1AP, 1:2500), HSPB5 (15808–1AP, 1:2500), SYNPO2 (Proteintech 25453–1AP, 1:2500), LC3 (Cell Signaling Technology 2775s, 1:1000). Secondary antibodies: LICOR IRDye 680RD and 800CW, 1:8000. Membranes were imaged on an Azure c600. Protein densitometry was quantified using ImageStudio (LI-COR Biosciences).

Martin T.G., Delligatti C.E., Muntu N.A., Stachowski-Doll M.J, & Kirk J.A. (2021). Pharmacological inhibition of BAG3-HSP70 with the proposed cancer therapeutic JG-98 is toxic for cardiomyocytes. Journal of cellular biochemistry, 123(1), 128-141.

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Immunoblotting of miRNA Targets and p27

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Cells were lysed in radioimmunoprecipitation (RIPA) buffer (50 mM Tris, pH 7.4, 1% TX-100, 0.2% Na deoxycholic acid, and 0.2% SDS, HALT complete tab [Roche]). Proteins were quantified using the Pierce BCA Protein Assay kit (ThermoFisher Scientific) and equal amounts of protein were run on 4–12% Bis-Tris gradient gels (Invitrogen) according to manufacturer’s instructions. Proteins were transferred by semi-dry electrophoresis (BioRad) onto Immobilon-P PVDF (EMD Millipore). Membranes were incubated overnight at 4°C with anti-p27 3688 (Cell Signaling Technologies) or HoxA7 ab70027 (Abcam), washed then incubated with anti-mouse or anti-rabbit HRP conjugated secondary antibodies (GE Healthcare). Membranes were developed on film with WesternSure Premium Chemiluminescent Substrate (LI-COR). The same Immunoblots were reprobed with β-actin clone AC-15 (Sigma) as loading control. Biotinylated miRNA target immunoblots and p27^Kip1 immunoblots were repeated on lysates generated from at least two independent experiments each.

Meyer S.E., Muench D.E., Rogers A.M., Newkold T.J., Orr E., O’Brien E., Perentesis J.P., Doench J.G., Lal A., Morris P.J., Thomas C.J., Lieberman J., McGlinn E., Aronow B.J., Salomonis N, & Grimes H.L. (2018). miR-196b target screen reveals mechanisms maintaining leukemia stemness with therapeutic potential. The Journal of Experimental Medicine, 215(8), 2115-2136.

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OXi6196-Induced Apoptosis Signaling

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MDA-MB-231 cells grown to 65–70% confluency were treated with 10 nM OXi6196 for varying times (0–90 min). Lysates were prepared by cold application of RIPA buffer, clarified via centrifugation, and treated with LDS/5% β-mercaptoethanol. Proteins were separated by SDS-PAGE on 4–12% Bis-Tris gradient gels (Invitrogen, ThermoFisher Scientific), then transferred to 0.2 μm PVDF membranes (EMD Millipore). After blocking, blots were incubated with primary antibodies (1:1000 for Cas-3, 1:2500 for GAPDH, Cell Signaling Technology), followed by secondary HRP-labeled goat anti-rabbit IgG antibodies (Jackson Immuno Research Labs, West Grove, PA USA). Blots were developed with Amersham ECL Prime chemiluminescent substrate and imaged with an ImageQuant LAS 4000 system (GE).

Liu L., Schuetze R., Gerberich J.L., Lopez R., Odutola S.O., Tanpure R.P., Charlton-Sevcik A.K., Tidmore J.K., Taylor E.A., Kapur P., Hammers H., Trawick M.L., Pinney K.G, & Mason R.P. (2022). Demonstrating Tumor Vascular Disrupting Activity of the Small-Molecule Dihydronaphthalene Tubulin-Binding Agent OXi6196 as a Potential Therapeutic for Cancer Treatment. Cancers, 14(17), 4208.

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Cell Lysis and Protein Extraction

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Cells were seeded into six-well plates at 50% of confluence, allowed to attach for 16 hr and treated. Cells were lysed in Cell Lysis Buffer (Cell Signaling Technology) containing Protease Inhibitor Cocktail (Roche Life Science, Indianapolis, IN) and PhosSTOP Phosphatase Inhibitor Cocktail (Roche). Lysates were centrifuged at 18,000 X g for 10 min at 4°C, and the supernatant was loaded to 4–12% Bis-Tris gradient gels (Invitrogen, Carlsbad, CA). The subsequent procedures were performed according to the recommendations of the antibody manufacturers.

Wang H.Q., Halilovic E., Li X., Liang J., Cao Y., Rakiec D.P., Ruddy D.A., Jeay S., Wuerthner J.U., Timple N., Kasibhatla S., Li N., Williams J.A., Sellers W.R., Huang A, & Li F. (2017). Combined ALK and MDM2 inhibition increases antitumor activity and overcomes resistance in human ALK mutant neuroblastoma cell lines and xenograft models. eLife, 6, e17137.

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Western Blot Protein Analysis

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Cells were washed once with 1X PBS and flash frozen in ethanol and dry ice. Cells were lysed in lysis buffer (50mM Tris pH 7.5, 100mM NaCl, 1% Triton X-100, 0.5% deoxycholate, .1% SDS). We measured protein concentration by a BCA assay (Pierce) and ran equal protein concentrations on 4–12% Bis-Tris gradient gels (Invitrogen). Protein was transferred to a PVDF membrane and incubated in blocking solution (5% nonfat dried milk, 0.1% Triton X-100, 1X PBS) for 2h at room temperature. Membranes were then incubated with primary antibody (in blocking solution) overnight at 4°C. Blots were washed 3X for 5m in washing solution (50mM Tris-Cl pH 7.5, 150mM NaCl, .05% Tween-20) and then incubated in blocking solution plus secondary antibody coupled to peroxidase (Sigma) for 1h. Membranes were washed 3X in washing solution and we detected protein levels with chemiluminescence (ECL Prime GE Healthcare). Molecular weights were identified using a protein standard (Precision Plus, Bio-Rad).

Paek A.L., Liu J.C., Loewer A., Forrester W.C, & Lahav G. (2016). Cell-to-cell variation in p53 dynamics leads to fractional killing. Cell, 165(3), 631-642.

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Archaella Glycoprotein Analysis via SDS-PAGE

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M. hungatei archaella were incubated for 1 h in 0.1% Triton X-100 (ref. 47 ), and subjected to SDS–PAGE using 4–12% Bis-Tris gradient gels (Invitrogen) according to the manufacturer’s instructions. Glycoprotein stains were performed using the Pro-Q Emerald 488 Glycoprotein Stain Kit from Thermo-Fischer (P21855) and incorporating CandyCane glycoprotein molecular weight standards (Molecular Probes) (Supplementary Fig. 7).

Poweleit N., Ge P., Nguyen H.H., Ogorzalek Loo R.R., Gunsalus R.P, & Zhou Z.H. (2016). CryoEM structure of the Methanospirillum hungatei archaellum reveals structural features distinct from the bacterial flagellum and type IV pili. Nature microbiology, 2, 16222.

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Cell Lysis and Western Blot Analysis

Cited 1 time

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Cells growing in monolayers were lysed using Cell Extraction Buffer (Life
Technologies) supplemented with complete protease inhibitors and PhosSTOP phosphatase
inhibitor cocktail tablets (Roche). Cell lysates were cleared by centrifugation, protein
concentrations were determined by DC Protein Assay (BioRad), and denatured lysates were
run on 4–12% Bis-Tris gradient gels (Invitrogen). Gels were transferred to
nitrocellulose membranes (BioRad) before being immunoblotted with indicated antibodies.
Cleaved-PARP antibody was obtained from Cell Signaling Technology. RAC1 activation assays
were performed as previously described according to the manufacturer's protocol (Cell
Biolabs) (10 (link)).

Watson I.R., Li L., Cabeceiras P.K., Mahdavi M., Gutschner T., Genovese G., Wang G., Fang Z., Tepper J.M., Stemke-Hale K., Tsai K.Y., Davies M.A., Mills G.B, & Chin L. (2014). The RAC1 P29S Hotspot Mutation in Melanoma Confers Resistance to Pharmacological Inhibition of RAF. Cancer research, 74(17), 4845-4852.

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Phospho-RPA Staining in Irradiated Cells

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For phospho-RPA staining, CH12 cells were left untreated, or were treated with 25 Gy of ionizing radiation using a Faxitron X-ray cabinet, and were then collected by centrifugation 3 h later. Pellets were lysed on ice for 10 min in high salt lysis buffer (50 mM Tris-HCl pH 7.6, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM DTT, 1x EDTA-free protease inhibitor cocktail (Roche, Basel, Switerzerland)), cleared by centrifugation at 20,000 x g for 10 min at 4 °C, and quantified by bicinchoninic acid assay (BCA; Pierce, Thermo Fisher Scientific). Equal amounts of whole-cell extracts were separated by SDS-PAGE on 4-12% Bis-Tris gradient gels (Invitrogen), transferred to nitrocellulose and immunoblotted for pRPA32 (S4/S8).

Noordermeer S.M., Adam S., Setiaputra D., Barazas M., Pettitt S.J., Ling A.K., Olivieri M., Álvarez-Quilón A., Moatti N., Zimmermann M., Annunziato S., Krastev D.B., Song F., Brandsma I., Frankum J., Brough R., Sherker A., Landry S., Szilard R.K., Munro M.M., McEwan A., Goullet de Rugy T., Lin Z.Y., Hart T., Moffat J., Gingras A.C., Martin A., van Attikum H., Jonkers J., Lord C.J., Rottenberg S, & Durocher D. (2018). The Shieldin complex mediates 53BP1-dependent DNA repair. Nature, 560(7716), 117-121.

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