Nupage novex 4 12 bis tris protein gel

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The NuPAGE® Novex® 4–12% Bis-Tris Protein Gels are pre-cast polyacrylamide gels designed for the separation and analysis of proteins. They feature a Bis-Tris buffer system and a 4-12% gradient, which allows for the effective separation of a wide range of protein sizes.

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123 protocols using nupage novex 4 12 bis tris protein gel

Western Blot Analysis Protocol

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For Western blots, proteins were run on NuPAGE™ Novex™ 4–12% Bis‐Tris Protein Gels (Thermo Fisher) before transferring to nitrocellulose membranes. For transfer, the iBlot system (Thermo Fisher) was used. Membranes were blocked using 5% milk powder (Marvel) or 0.125% BSA (Sigma) and 0.125% milk powder (Marvel) in TBS containing 0.1% Tween‐20 (TBST) for 30 min at room temperature then incubated with primary antibody at 4°C overnight. HRP‐conjugated secondary antibodies (Thermo Fisher) diluted 1:10,000 in blocking buffer were incubated with the blots for 1 h at room temperature. Chemiluminescence was detected in a Bio‐Rad chemidoc using Immobilon reagent (Millipore). Protein loading was checked by staining gels with Colloidal Coomassie Blue Stain (Severn Biotech). Densitometric analysis was carried out using Image Lab 4.1 (Bio‐Rad Laboratories).

Wong D.C., Seinkmane E., Zeng A., Stangherlin A., Rzechorzek N.M., Beale A.D., Day J., Reed M., Peak‐Chew S.Y., Styles C.T., Edgar R.S., Putker M, & O’Neill J.S. (2021). CRYPTOCHROMES promote daily protein homeostasis. The EMBO Journal, 41(1), e108883.

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Western Blot Analysis of Protein Expression

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Cells were lysed in LDS sample buffer, 50 μg total protein was separated on NuPAGE Novex 4–12% Bis-Tris Protein Gels (ThermoFisher Scientific) and transferred to 0.2 μm nitrocellulose membranes (GE Healthcare, Vienna, Austria). The following antibodies were used: anti-MAO-A rabbit Mab (EPR7101, 1:2000) (Abcam), anti-E-cadherin (Cat:610181, 1:500) (Becton Dickinson, Heidelberg, Germany), Vimentin (SC6260, 1:500) (Cruz Biotechnology, Santa Cruz, CA), anti-GAPDH (MAB374, 1:50.000) (Millipore, Vienna, Austria), anti cPARP (G7341, 1:500) (Sigma), and anti-p21 (2946 S, 1:500) (Cell Signaling, Frankfurt, Germany). Specificity of the used MAO-A antibody was tested and confirmed using protein of PF179TCAF cells after treatment with either 100 nM Dex, or a combination of 100 nM Dex and 6 µM RU486 for 3 days and using protein of PC3 cells overexpressing MAO-A. Furthermore, reduced MAO-A protein was confirmed after transient MAO-A knockdown for 3 days in LNCaPabl cells utilizing ON-TARGET plus Human MAO-A siRNA SMARTpool (L-009369-00-0005), siControl On-Target plus Non-targeting pool (D-001810-10-05) and Lipofectamine2000 (ThermoFisher Scientific) according to the manufacturer’s protocols.

Puhr M., Eigentler A., Handle F., Hackl H., Ploner C., Heidegger I., Schaefer G., Brandt M.P., Hoefer J., Van der Pluijm G, & Klocker H. (2021). Targeting the glucocorticoid receptor signature gene Mono Amine Oxidase-A enhances the efficacy of chemo- and anti-androgen therapy in advanced prostate cancer. Oncogene, 40(17), 3087-3100.

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Western Blot Analysis of Protein Samples

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Cell lysates were prepared in RIPA buffer supplemented with proteinase and phosphatase inhibitors. Proteins were resolved on NuPAGE Novex 4–12% Bis-Tris Protein Gels (Thermo Fisher Scientific) and transferred electronically onto a PVDF 0.45 μM membrane (Millipore). Membranes were blocked in 5% BSA diluted in Tris buffer saline plus 0.1% Tween 20 (TBST) for 1 hour at room temperature and were incubated with primary antibodies in 5% BSA at 4 °C overnight. After 3 washes of 10 min TBST, membranes were incubated with secondary antibodies in 5% BSA diluted in TBST for 1 hour at room temperature. After 3 washes of 10 min in TBST, membranes were developed using an Enhanced Chemiluminescence (ECL) kit (GE Healthcare Life Sciences).

Mao N., Gao D., Hu W., Hieronymus H., Wang S., Lee Y.S., Lee C., Choi D., Gopalan A., Chen Y, & Carver B.S. (2019). Aberrant expression of ERG promotes resistance to combined PI3K and AR pathway inhibition through maintenance of AR target genes. Molecular cancer therapeutics, 18(9), 1577-1586.

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Phosphorylation of Bub1 by CDK1-CyclinB1 and Mps1

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Recombinant GST-Bub1 (12 μg) was incubated with 20 units CDK1-CyclinB1 (New England Biolabs), 0,2 μg Mps1 (TTK; Life Technologies) in protein kinase buffer (50 mM Tris pH 7.5, 10 mM MgCl₂, 0.1 mM EDTA, 2 mM DTT, 0.01% Brij 35). 500 μM ATP and 125 nM Calyculin A (Cell Signaling) were added and the reactions were incubated at 30 °C for 1 h. Controls were incubated in kinase buffer and Calyculin A only. 6 μg of phosphorylated protein was resolved on NuPAGE Novex 4–12% Bis-Tris protein gels, which were fixed and stained by Colloidal Blue Staining (Thermo Scientific). The band corresponding to GST-Bub1 was cut out and analysed by mass spectrometry.

Zhang G., Kruse T., López-Méndez B., Sylvestersen K.B., Garvanska D.H., Schopper S., Nielsen M.L, & Nilsson J. (2017). Bub1 positions Mad1 close to KNL1 MELT repeats to promote checkpoint signalling. Nature Communications, 8, 15822.

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Immunoblotting Analysis of Yeast Proteins

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Proteins were resolved by gel electrophoresis using NuPAGE Novex 4–12% Bis-Tris Protein Gels (Thermo Fisher Scientific). Proteins were transferred to nitrocellulose and bands detected by immunoblotting with specific rabbit polyclonal antisera. Antibodies against Sec61p, Sbh1p, CPY [29 (link)], Rpn12 [16 (link)], and preproalpha factor (ppαF) [29 (link)] had been previously raised in our laboratory and were used at a dilution of 1:2000; antibodies against Sss1p and Sec63p were kindly provided by Randy Schekman and used at a dilution of 1:2500. Signals were developed by enhanced chemiluminescence using the SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) according to the manufacturer´s instructions.

Elia F., Yadhanapudi L., Tretter T, & Römisch K. (2019). The N-terminus of Sec61p plays key roles in ER protein import and ERAD. PLoS ONE, 14(4), e0215950.

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Western Blot Analysis of Telomerase Protein

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Cells were lysed in RIPA buffer (89901, ThermoFisher, Waltham, MA) supplemented with 1× protease inhibitor cocktail (PI78410, ThermoFisher, Waltham, MA) and then mixed with 4x LDS sample buffer (NP0008, ThermoFisher, Waltham, MA) and boiled at 95° C for 10 minutes. Samples were run on NuPAGE Novex 4–12% Bis-Tris Protein Gels (NP0335, ThermoFisher, ThermoFisher Scientific), and transferred to PVDF membranes. Membranes were incubated overnight at 4C with rabbit anti-human Tert antibody, (1:100 dilution) (Y182 ab32020, Abcam, Cambridge, MA) then HRP-linked anti-rabbit IgG antibody (1:2000 dilution) (7074S, Cell Signaling Technology, Danvers, MA) and visualized with Pierce ECL Western Blotting Substrate (32106, ThermoFisher, Waltham, MA) on the Bio-Rad ChemiDoc XRS+ System.

Guzman H., Sanders K., Idica A., Bochnakian A., Jury D., Daugaard I., Zisoulis D.G, & Pedersen I.M. (2018). miR-128 inhibits telomerase activity by targeting TERT mRNA. Oncotarget, 9(17), 13244-13253.

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Extracting and Quantifying Proteins by Western Blot

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Total proteins were extracted with RIPA lysis buffer (89900, Thermo Scientific) supplemented with 1X Protease Inhibitor—Complete ULTRA tablets mini (5892791001, Roche) and 1X Benzonase nuclease HC (712063, Millipore) for 1 h at 4°C. Equal amounts of total cellular proteins were resolved on NuPAGE® Novex® 4–12% Bis-Tris Protein Gels (NP0336BOX, Thermo Fisher Scientific) and transferred to nitrocellulose membranes (iBlot, Thermo Fisher Scientific) following the manufacturer’s instructions. Membranes were then blocked in Odyssey Blocking Buffer (927-4-0000, Li-Cor) for 1 h at room temperature. Incubation with primary antibodies was carried out at 4°C overnight in Odyssey Blocking Buffer. The following antibodies were used: rabbit anti-β-SG (dilution 1/100, HPA011422, Sigma) and rabbit anti-Actinin alpha (H-300) (dilution 1/1000, sc-15335, Santa Cruz). After 1 h incubation with donkey anti-rabbit 680 antibody (926–68073, EuroBio) at room temperature, proteins were detected by fluorescence (Odyssey, Li-Cor) following the manufacturer’s instructions. Western blot signal quantification was performed with the Plot Lanes Analysis tool of Image J software (NIH).

Henriques S.F., Patissier C., Bourg N., Fecchio C., Sandona D., Marsolier J, & Richard I. (2018). Different outcome of sarcoglycan missense mutation between human and mouse. PLoS ONE, 13(1), e0191274.

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Adipose and Liver Protein Extraction and Western Blotting

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Proteins were extracted from adipose tissue or liver samples with NP-40 lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 10 mM NaF, 2 mM Na₃VO₄, PMSF) with complete protease inhibitors (Sigma-Aldrich) and subjected to 4–12% gradient SDS-polyacrylamide gel electrophoresis (NuPAGE™ Novex™ 4–12% Bis-Tris Protein Gels, ThermoFisher). Proteins from PAGE gels were transferred to PVDF membranes. Membranes were blotted for FABP5 (Cell Signaling #39926, 1:1000), FABP4 (inhouse, clone HA3 (Burak et al., 2015 (link)) HRP conjugated, 1:10,000; or Proteintech #12802-1-AP, 1:5,000 in FABP secretion assay), ACC (Cell Signaling #3662, 1:1000; or #3676, 1:5,000 in adipose explant analysis), ACLY (Cell Signaling #4332, 1:1000, or 1:5,000 in adipose explant analysis), ACAS2 (Cell Signaling #3658, 1:5,000), SREBP1 (Santa Cruz, clone K-10, sc-367, 1:500), ChREBP (Santa Cruz, clone M-300, sc-33764, 1:500), GAPDH (Bioeasytech #BE0023, 1:5,000), HSP70 (Proteintech, #10995-1-AP, 1:5,000), and beta-Tubulin (Santa Cruz, sc-9104, 1:1000).

Charles K.N., Li M.D., Engin F., Arruda A.P., Inouye K, & Hotamisligil G.S. (2017). Uncoupling of metabolic health from longevity through genetic alteration of adipose tissue lipid binding proteins. Cell reports, 21(2), 393-402.

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Western Blotting of Whole Cell Lysates

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HeLa cells were lysed in lysis buffer [20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% NP40 (or Triton-X-100), pH 7.4 plus protease inhibitor cocktail (Calbiochem set I) and phosphatase inhibitor cocktail (Calbiochem set II)]; lysates were fractionated by SDS-PAGE on 10% gels under reducing conditions and immunoblotted onto nitrocellulose membranes. Bands were detected by using enhanced chemiluminescence (Pierce), and exposed films developed on a SRX-101A Film Processor (Konica).
SCC47 cells were lysed with CellLytic M (Sigma-Aldrich) with complete Mini Protease Inhibitor Cocktail tablets (Sigma-Aldrich) and phosphatase inhibitors PhosSTOP (Sigma-Aldrich). Lysates were fractionated by SDS-PAGE on NuPage Novex 4–12% Bis-Tris protein gels (Thermo Fisher) and immunoblotted onto Immobilon-FL PVDF membrane (Millipore). Proteins were detected using the Odyssey Imaging System (Li-Cor).

Tomas A., Jones S., Vaughan S.O., Hochhauser D, & Futter C.E. (2017). Stress-specific p38 MAPK activation is sufficient to drive EGFR endocytosis but not its nuclear translocation. Journal of Cell Science, 130(15), 2481-2490.

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Western Blot Analysis of Geminin and HA-tagged Proteins

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Cells were lysed using RIPA lysis buffer with Halt Protease and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific). Protein concentration was determined using the Bradford protein assay, and 20 μg of samples were loaded along with Nupage LDS Sample Buffer 4x (ThermoFisher Scientific) onto NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gels (ThermoFisher Scientific). The protein was transferred onto a 0.45μm nitrocellulose membrane using a Trans-Blot Turbo Transfer System (Bio-Rad). Immunoblotting was performed with the following antibodies: 1/1000 anti-Geminin (Abcam ab12147), 1/1000 anti-HA (Covance MMS-101P), 1/2000 anti-Beta Actin HRP (CST 5125S), 1/1000 anti-mouse HRP (MP Biomedicals), 1/1000 anti-rabbit HRP (Jackson ImmunoResearch).

Tan J, & Martin S.E. (2016). Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening. PLoS ONE, 11(12), e0168968.

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