Bis tris protein gel

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

Bis-Tris protein gels are electrophoresis gel products designed for the separation and analysis of proteins. They utilize a Bis-Tris buffer system to provide effective separation and resolution of protein samples.

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NuPAGE 4–12% Bis-Tris proteins gels 4 to 12% Bis‐Tris protein gradient gels Novex NuPAGE 4–12% Bis-Tris protein gels NuPAGE 4 to 12% gradient Bis-Tris polyacrylamide protein gels Novex 4–16% Bis-Tris Protein Gels NuPAGE Bis-Tris 4–12% Protein Gels NuPAGE™ Bis-Tris Mini Protein Gels Bolt 8% Bis-Tris plus 1.0 mm 12 well precast protein gels NuPAGE® 4–12 % Bis-Tris Precast Protein Gels NativePAGE Novex 3–12% Bis-Tris Protein Gels

111 protocols using bis tris protein gel

Protein Extraction and Western Blot Analysis

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Protein was collected using RIPA Lysis and Extraction buffer
(ThermoScientific) with PhosSTOP phosphatase inhibitor (Roche) and cOmplete
ULTRA mini protease inhibitor (Roche). 20μg of protein was separated
on a 4–12% Bis-Tris protein gel (Novex), transferred using
the iBlot2 Gel Transfer device (ThermoScientific) onto a nitrocellulose
membrane (Novex) gel, blocked with Rockland Blocking Buffer (Rockland) and
incubated overnight at 4°C with primary antibodies anti-TREX1
(Abcam, ab185228, 1:1000), anti-IRF3 (Cell Signaling,
4302S, 1:1000), anti-phospho-IRF3 (Cell Signaling,
4947S, 1:1000), anti-LINE-1ORF1p (Millipore,
MABC1152, 1:1000), and anti- β-Actin (Abcam,
ab8226, 1:7000), diluted in 3% BSA. The
following day, the cells were incubated with the secondary antibodies for
two hours at room temperature and imaged and quantified using the Odyssey
CLx imaging system (Licor).

Thomas C.A., Tejwani L., Trujillo C.A., Negraes P.D., Herai R.H., Mesci P., Macia A., Crow Y.J, & Muotri A.R. (2017). Modeling of TREX1-dependent autoimmune disease using human stem cells highlights L1 accumulation as a source of neuroinflammation. Cell stem cell, 21(3), 319-331.e8.

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Protein Lysate Preparation and Western Blotting

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RIPA buffer (Sigma‐Aldrich) with 1mM PMSF (Sigma‐Aldrich), 5mM Z‐VAD (Promega), 1mM NaVan (Sigma‐Aldrich), and 1x Complete Protease Inhibitor Cocktail tablets (Roche) was used to prepare Protein lysate of half brain and CD11b⁺ cells lysis from mice. For western blotting, 30 µg of protein lysate was separated on 12% Bis‐Tris protein gel (Novex) with 20X MES running buffer (Novex) and transferred on nitrocellulose membranes. Primary antibodies (listed in Table S5) were incubated at 4°C overnight. Secondary antibodies (1:10,000) were used: Alexa‐Fluor goat anti‐rabbit 800, Alexa‐Fluor goat anti‐mouse 680, Alexa‐Fluor goat anti‐rabbit 680, and Alexa‐Fluor goat anti‐rat 800 (all from Life Technologies). The membrane was imaged using the LiCor Imaging System and Odyssey V3.0 software (LiCor), followed by intensity analysis with ImageJ.

Ziaei A., Garcia‐Miralles M., Radulescu C.I., Sidik H., Silvin A., Bae H., Bonnard C., Yusof N.A., Ferrari Bardile C., Tan L.J., Ng A.Y., Tohari S., Dehghani L., Henry L., Yeo X.Y., Lee S., Venkatesh B., Langley S.R., Shaygannejad V., Reversade B., Jung S., Ginhoux F, & Pouladi M.A. (2022). Ermin deficiency leads to compromised myelin, inflammatory milieu, and susceptibility to demyelinating insult. Brain Pathology, 32(5), e13064.

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Quantitative Western Blot Analysis

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Protein was extracted using the RIPA Lysis and Extraction buffer (Thermo Fisher) containing cOmplete ULTRA mini protease inhibitor (Roche) and PhosSTOP phosphatase inhibitor (Roche). Twenty microgram of protein lysates were separated on a 4–12% Bis-Tris protein gel (Novex), and transferred onto a nitrocellulose membrane (Novex) using the iBlot2 Gel Transfer device (Thermo Fisher). Following blockage with Rockland Blocking Buffer (Rockland), the membrane was incubated with primary antibodies overnight at 4 °C. Next, the membrane was washed five times (5 min each) with 0.1% Tween 20 in PBS and incubated with secondary antibodies for 2 h at room temperature. Antibodies used in this study can be found in Table S9. Odyssey CLx imaging system (Li-Cor) was used for signal detection, and semi-quantitative analysis was performed using Odyssey Image Studio software.

Negraes P.D., Trujillo C.A., Yu N.K., Wu W., Yao H., Liang N., Lautz J.D., Kwok E., McClatchy D., Diedrich J., de Bartolome S.M., Truong J., Szeto R., Tran T., Herai R.H., Smith S.E., Haddad G.G., Yates JR 3.r.d, & Muotri A.R. (2021). Altered network and rescue of human neurons derived from individuals with early-onset genetic epilepsy. Molecular Psychiatry, 26(11), 7047-7068.

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Protein Extraction and Western Blot

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Protein was extracted using RIPA Lysis and Extraction buffer (Thermo Fisher) containing cOmplete ULTRA mini protease inhibitor (Roche) and PhosSTOP phosphatase inhibitor (Roche). Twenty μg of protein lysates were separated on a 4%–12% Bis-Tris protein gel (Novex), and transferred onto a nitrocellulose membrane (Novex) using the iBlot2 Gel Transfer device (Thermo Fisher). Following blockage with Rockland Blocking Buffer (Rockland), the membrane was incubated with primary antibodies overnight at 4°C. Next, the membrane was washed five times (5 min each) with 0.1% Tween 20 in PBS and incubated with secondary antibodies for 2 h at room temperature. Antibodies used in this study can be found in Supplementary Table S9. Odyssey CLx imaging system (Li-Cor) was used for signal detection and semi-quantitative analysis was performed using Odyssey Image Studio software.

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Western Blot Analysis of Skin Proteins

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Western blot analysis was performed, as described previously [33 (link)]. Briefly, cell lysates, prepared in radioimmunoprecipitation assay buffer (RIPA Lysis and Extraction Buffer), were resolved by electrophoresis on 4–12% Bis-Tris protein gel (Invitrogen, Waltham, MA, USA) under denaturing conditions using SDS. All procedures are conducted following the manufacturer’s instructions. Resultant bands were blotted onto polyvinylidene difluoride membranes, probed with anti-human keratin 10 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-human involucrin Abcam (Cambridge, UK), or anti-human β-actin (Sigma-Aldrich), and detected using enhanced chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA). The intensity of bands was measured with a LAS-3000 (Fujifilm, Tokyo, Japan).

Shin K.O., Mihara H., Ishida K., Uchida Y, & Park K. (2022). Exogenous Ceramide Serves as a Precursor to Endogenous Ceramide Synthesis and as a Modulator of Keratinocyte Differentiation. Cells, 11(11), 1742.

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Immunoblot Analysis of AcrIIA4 Expression

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AcrIIA4 was immunoblotted with K562 cells expressing either control (untagged AcrIIA4) or AcrIIA4-3XFLAG. Whole-cell extracts were prepared from 1× radioimmunoprecipitation assay lysis buffer (Millipore). Extracts were clarified by centrifugation at 15,000g for 15 min at 4°C, and protein concentrations were determined by Pierce BCA (bicinchoninic acid) assay (Thermo Fisher Scientific). Eight micrograms of whole-cell extract was separated on precast 4 to 12% bis tris protein gel (Invitrogen) and transferred to a nitrocellulose membrane. Membranes were blocked in PBS–0.05% Tween 20 (PBST) containing 5% nonfat dry milk and incubated overnight at 4°C with primary antibody [FLAG M2 (F1804, Sigma) and glyceraldehyde-3-phosphate dehydrogenase (14C10, Cell Signaling)] diluted in PBST–5% nonfat dry milk. Membranes were subsequently washed with PBST and incubated with the appropriate IRDye 680RD and IRDye 800CW secondary antibody (LI-COR Biosciences) diluted in PBST–5% nonfat dry milk. Images were detected using the Odyssey Systems (LI-COR Biosciences).

Shin J., Jiang F., Liu J.J., Bray N.L., Rauch B.J., Baik S.H., Nogales E., Bondy-Denomy J., Corn J.E, & Doudna J.A. (2017). Disabling Cas9 by an anti-CRISPR DNA mimic. Science Advances, 3(7), e1701620.

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Quantifying Apoptosis-Related Protein Levels

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Protein levels of BAX, BCL-2, caspase 3 and β-actin were assessed using Western blot analysis, as described previously [26 (link),27 (link)]. Briefly, cell lysates, prepared in radioimmunoprecipitation assay buffer, were resolved by electrophoresis on 4–12% Bis-Tris protein gel (Invitrogen). Resultant bands were blotted onto polyvinylidene difluoride membranes, probed with anti-BAX, anti- BCL-2, anti-caspase 3(Santa Cruz Biotechnology, Dallas, TX, USA), and anti-human β-actin (Sigma-Aldrich, St. Louis, MO, USA), and detected using enhanced chemiluminescence (Thermo Fisher Scientific). Band intensity was measured with a LAS-3000 (Fujifilm, Tokyo, Japan).

Shin K.O., Kim S., Kim B., Park H.Y., Jung E., Kim G., Kim D., Cho H.E., Uchida Y, & Park K. (2022). Euphorbia supina Extracts Block NADPH Oxidase-Mediated, Ceramide-Induced Apoptosis Initiated by Diesel Particulate Matter. Pharmaceuticals, 15(4), 431.

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Western Blot Analysis of ZO-1 in Caco-2 Cells

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Caco-2 cells were washed twice with PBS and subsequently lysed. Intestinal tissue samples were homogenized in protein lysis buffer and subsequently sonicated. Protein extracts were separated on 8% SDS-polyacrylamide or 4–12% bis−tris protein gel (Invitrogen), transferred on a nitrocellulose membrane, and stained with antibodies against ZO-1 (N1N2, GeneTex, dilution 1:3000). An antibody against β-actin (AC-15, abcam, dilution 1:25,000) was used as loading control. Supplementary Fig. 10 shows the uncropped gel scans of Western blots.

Tajik N., Frech M., Schulz O., Schälter F., Lucas S., Azizov V., Dürholz K., Steffen F., Omata Y., Rings A., Bertog M., Rizzo A., Iljazovic A., Basic M., Kleyer A., Culemann S., Krönke G., Luo Y., Überla K., Gaipl U.S., Frey B., Strowig T., Sarter K., Bischoff S.C., Wirtz S., Cañete J.D., Ciccia F., Schett G, & Zaiss M.M. (2020). Targeting zonulin and intestinal epithelial barrier function to prevent onset of arthritis. Nature Communications, 11, 1995.

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Mouse Protein Extraction and Immunoblotting

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Tissues were collected to prepare protein extracts from C57BL/6 mice and lysed in TAP lysis buffer containing 50 mmol/L HEPES–KOH, pH 7.5, 100 mmol/L KCl, 2 mmol/L EDTA, 10% glycerol, 0.1% NP-40, 10 mmol/L NaF, 0.25 mmol/L Na₃VO₄ and 50 mmol/L β-glycerolphosphate plus protease inhibitors (Roche, 04693132001). After homogenization, the cell extracts were placed on ice stand for 30 min, followed by centrifugation at 4°C at 13,000 ×g for 20 min. The supernatant was extracted for immunoblotting assays. Equal amounts of proteins were electrophoretic on a 10% BIS-TRIS protein gel (Invitrogen, NP0315) and the bands were transferred to a polyvinylidene fluoride film (Millipore). The primary antibodies for immunoblotting included RBM46 (1:500 dilution; Diaan Wuhan, customed), RAD21 (1:500 dilution; Abclonal, A18850), and GAPDH (1:5,000 dilution; Abways, AB0037).

Lv Y., Lu G., Cai Y., Su R., Liang L., Wang X., Mu W., He X., Huang T., Ma J., Zhao Y., Chen Z.J., Xue Y., Liu H, & Chan W.Y. (2022). RBM46 is essential for gametogenesis and functions in post-transcriptional roles affecting meiotic cohesin subunits. Protein & Cell, 14(1), 51-63.

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Quantifying MMP-1 and MMP-3 Protein Levels

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Protein levels of MMP-1 and MMP-3 were assessed by Western blot analysis, as we described previously [40 (link)]. Briefly, cell lysates, prepared in radioimmunoprecipitation assay buffer, were resolved by electrophoresis on 4–12% Bis-Tris protein gel (Invitrogen, Carlsbad, CA, USA). Resultant bands were blotted onto polyvinylidene difluoride membranes, probed with anti-MMP-1 and MMP-3 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-phospho-STAT1 (Tyr-701) anti-phospho-STAT3 (Tyr-705) (Cell Signaling Technology, Boston, MA, USA), and anti-human β-actin (Sigma-Aldrich), and detected using enhanced chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA). The intensity of bands was measured with a LAS-3000 (Fujifilm, Tokyo, Japan).

Shin K.O., Uchida Y, & Park K. (2022). Diesel Particulate Extract Accelerates Premature Skin Aging in Human Fibroblasts via Ceramide-1-Phosphate-Mediated Signaling Pathway. International Journal of Molecular Sciences, 23(5), 2691.

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