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Bis tris midi gel

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The Bis-Tris Midi Gel is a pre-cast polyacrylamide gel used for electrophoresis. It is designed to provide effective separation of proteins in the molecular weight range of 10-200 kDa.

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Lab products found in correlation

Reducing agent, by Thermo Fisher Scientific (3 mentions) Qubit protein assay kit, by Thermo Fisher Scientific (3 mentions) Iblot dry blotting system, by Thermo Fisher Scientific (3 mentions) Anti actin, by Merck Group (3 mentions) Qubit fluorometer, by Thermo Fisher Scientific (3 mentions) Odyssey clx, by LI COR (2 mentions) Caspase 8, by Cell Signaling Technology (2 mentions) Iblot gel transfer stacks pvdf regular, by Thermo Fisher Scientific (2 mentions) Chameleon duo pre stained protein ladder, by LI COR (2 mentions) Bca reagent, by Thermo Fisher Scientific (2 mentions) β actin, by Merck Group (2 mentions) Odyssey blocking buffer tbs, by LI COR (2 mentions) Hrp linked antibody goat anti rabbit, by Merck Group (2 mentions) Anti arc, by Synaptic Systems (2 mentions) Image station 440cf, by Kodak (2 mentions) Iblot system, by Thermo Fisher Scientific (2 mentions) Iblot gel transfer system, by Thermo Fisher Scientific (1 mentions) Ab6795, by Abcam (1 mentions) D6w5b, by Cell Signaling Technology (1 mentions) Ab125066, by Abcam (1 mentions)

Spelling variants of this product

Bis-Tris gels (1248 citations) NuPAGE Novex Bis-Tris (4%–12%) midi gels (8 citations) NuPAGE™ 4–12% Bis-Tris Midi Protein Gels (5 citations) Novex 4-12% Bis-Tris Midi protein gels (2 citations) NuPAGE® Novex Bis‐Tris Midi‐ Gels (2 citations) Novex Bis-Tris Midi gradient gels (2 citations) NuPAGE Novex 4–12% bis-tris midi protein gels (2 citations) Novex Bis-Tris Midi gel 1.0 mm precast gels (2 citations)

18 protocols using bis tris midi gel

1

Western Blot Analysis of Cell Signaling

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Cells were lysed in RIPA buffer, with protease/phosphatase inhibitors, and sonicated to extract protein. Protein concentration was measured using the BCA reagent (Thermo Fisher Scientific) and protein samples were run on NUPAGE 4%–12% Bis-Tris Midi Gels (Life Technologies, NP0321) along with 10 μL of Chameleon Duo Pre-stained protein ladder (LI-COR, 928-60000). The protein was transferred to a nitrocellulose membrane using the iBlot Transfer machine and iBlot gel transfer Stacks PVDF Regular (Life Technologies, 1340109). The membranes were blocked in Odyssey TBS blocking buffer (LI-COR, 927-50000) containing 0.1% Tween 20 for 1 hour at room temperature (RT). Membranes were incubated overnight at 4°C with the following primary antibodies: pRIPK1 (phospho-S166; 1:1000; Cell Signaling #31122 and #65746), RIPK1 (1:1000; BD Biosciences #610459 and Cell Signaling #3493), pMLKL (phospho-S345; 1:1000; abcam #ab196436 and phospho-S358; 1:1000; Cell Signaling #91689), MLKL (1:1000; Cell Signaling #14993 and #37705), MBP (1:1000; abcam ab62631), caspase-8 (1:1000; Cell Signaling #9746), and β-actin (1:5,000; Sigma-Aldrich #A5441). Membranes were incubated with LI-COR secondary antibodies at a 1:5000 dilution, imaged on the LI-COR Odyssey CLx and quantified with FIJI.
Zelic M., Pontarelli F., Woodworth L., Zhu C., Mahan A., Ren Y., LaMorte M., Gruber R., Keane A., Loring P., Guo L., Xia T.H., Zhang B., Orning P., Lien E., Degterev A., Hammond T, & Ofengeim D. (2021). RIPK1 activation mediates neuroinflammation and disease progression in multiple sclerosis. Cell reports, 35(6), 109112.
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2

Western Blot Analysis of Cell Signaling

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Cells were lysed in RIPA buffer, with protease/phosphatase inhibitors, and sonicated to extract protein. Protein concentration was measured using the BCA reagent (Thermo Fisher Scientific) and protein samples were run on NUPAGE 4%–12% Bis-Tris Midi Gels (Life Technologies, NP0321) along with 10 μL of Chameleon Duo Pre-stained protein ladder (LI-COR, 928-60000). The protein was transferred to a nitrocellulose membrane using the iBlot Transfer machine and iBlot gel transfer Stacks PVDF Regular (Life Technologies, 1340109). The membranes were blocked in Odyssey TBS blocking buffer (LI-COR, 927-50000) containing 0.1% Tween 20 for 1 hour at room temperature (RT). Membranes were incubated overnight at 4°C with the following primary antibodies: pRIPK1 (phospho-S166; 1:1000; Cell Signaling #31122 and #65746), RIPK1 (1:1000; BD Biosciences #610459 and Cell Signaling #3493), pMLKL (phospho-S345; 1:1000; abcam #ab196436 and phospho-S358; 1:1000; Cell Signaling #91689), MLKL (1:1000; Cell Signaling #14993 and #37705), MBP (1:1000; abcam ab62631), caspase-8 (1:1000; Cell Signaling #9746), and β-actin (1:5,000; Sigma-Aldrich #A5441). Membranes were incubated with LI-COR secondary antibodies at a 1:5000 dilution, imaged on the LI-COR Odyssey CLx and quantified with FIJI.
Zelic M., Pontarelli F., Woodworth L., Zhu C., Mahan A., Ren Y., LaMorte M., Gruber R., Keane A., Loring P., Guo L., Xia T.H., Zhang B., Orning P., Lien E., Degterev A., Hammond T, & Ofengeim D. (2021). RIPK1 activation mediates neuroinflammation and disease progression in multiple sclerosis. Cell reports, 35(6), 109112.
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3

Western Blot Protein Analysis

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was performed by the addition of 10 μg of protein from the stem cell lysates, determined by a modified Bradford assay, to the lanes of 4–12% Bis-Tris Midi gels (Life Technologies). The proteins were separated by electrophoresis, then transferred to a nitrocellulose membrane using the iBlot gel transfer system (Life Technologies). The nitrocellulose membrane was blocked with tris-buffered saline with 0.05% TWEEN 20 and 5% w/v non-fat dried skimmed milk, prior to probing with primary antibodies EML4 (HPA036688 Sigma Aldrich) and GAPDH, labelled with HRP (sc-25778 HRP Santa-Cruz), all at 1:4000 dilutions. The anti-EML4 antibody was detected with sheep anti-rabbit antibody, labelled with HRP (ab6795 Abcam). The substrate ECL 2 (Fisher Scientific) was added, and the membranes were exposed to FujiFilm Super RX films (Fisher Scientific) prior to development in Kodak GBX developer and fixer solutions.
Madgwick A., Fort P., Hanson P.S., Thibault P., Gaudreau M.C., Lutfalla G., Möröy T., Abou Elela S., Chaudhry B., Elliott D.J., Morris C.M, & Venables J.P. (2015). Neural Differentiation Modulates the Vertebrate Brain Specific Splicing Program. PLoS ONE, 10(5), e0125998.
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4

Western Blot Analysis of Protein Markers

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Protein extracts were separated by 1D gel electrophoresis using NuPAGE 4–12% (w/v) Bis-Tris midi gels (Invitrogen, USA), and transferred onto PVDF membranes using an iBlot 2 transfer system (Invitrogen, USA), according to the manufacturer’s specifications. Immunoblots were performed to detect FLAG tag (1:1000, D6W5B, Cell Signaling Technology, USA), LRP8/APOER2 (1:2000; ab108208, Abcam, UK), β-ACTIN (1:5000, AC-15, Sigma-Aldrich, Australia), GPX4 (1:1000, ab125066, Abcam, USA), Notch1 (1:1000, D1E11, Cell Signaling Technology, USA), PS1 (1:1000, D39D1, Cell Signaling Technology, USA), or PS2 (1:1000, D30G3, Cell Signaling Technology, USA), SELENOP (B-9, Mouse mAb; sc-376858, Santa Cruz Biotechnology, Inc., USA), cleaved caspase 3 (Asp175; 1:1000, 5A1E, Rabbit mAb 9664; Cell Signaling Technology, USA), or cleaved caspase 7 (Asp198; 1:1000, D6H1, Rabbit mAb 8438) followed by appropriate HRP-conjugated secondary antibodies (1:5000, Thermo, Australia). Chemiluminescence was detected using Pierce ECL (Thermo, Australia) and visualized using the LAS-3000 Imaging System (Fujifilm, Japan) or Odyssey® Fc Imaging System (LI-COR Biosciences, Lincoln, NE). Representative blots for all proteins (including β-actin) are shown. Original western blots for all relevant figures are shown in “Supplementary Material—Original Blots”.
Greenough M.A., Lane D.J., Balez R., Anastacio H.T., Zeng Z., Ganio K., McDevitt C.A., Acevedo K., Belaidi A.A., Koistinaho J., Ooi L., Ayton S, & Bush A.I. (2022). Selective ferroptosis vulnerability due to familial Alzheimer’s disease presenilin mutations. Cell Death and Differentiation, 29(11), 2123-2136.
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5

Verifying Immune Protein Expression

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Two of the identified immune-related proteins, Cyclophilin A (CypA) and Cofilin 1 (Cof1), that were included in the list of proteins contributing to the separation of the TDI-treated group and AOO-treated group in the PCA were verified via Western blotting in B lymphocytes obtained from a new, independent set of similarly treated TDI- and AOO-treated mice (n = 10/group). Briefly, proteins were loaded and separated on 4–12% Bis/Tris Midi-gels (Invitrogen, Merelbeke, Belgium) and subsequently transferred to a PVDF membrane (iBlot, Gel Transfer Stack, Invitrogen). Membranes were blocked (1–2 h, 5% blocking agent, GE Healthcare) and incubated overnight with primary antibody (LSP-1: 1/1000, goat Ab, Santa Cruz Biotechnology; CypA: 1/1000, mouse Ab, Santa Cruz Biotechnology; GAPDH, internal standard, 1/200000, mouse Ab, Dako). Following secondary Ab incubation (LSP-1: 1/50000, donkey anti-goat IgG, Santa Cruz Biotechnology; Cof1 and GAPDH: 1/100000, goat anti mouse IgG, Dako) protein bands were visualized using chemiluminescence detection (Supersignal West Dura, Thermo Scientific) on ECL hyperfilm (GE Healthcare). The protein bands were semiquantitatively evaluated by densitometry (ImageQuant TL v2009, GE Healthcare).
Haenen S., Vanoirbeek J.A., De Vooght V., Schoofs L., Nemery B., Clynen E, & Hoet P.H. (2015). Proteomic Alterations in B Lymphocytes of Sensitized Mice in a Model of Chemical-Induced Asthma. PLoS ONE, 10(9), e0138791.
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6

Quantitation of Mutant Huntingtin Protein

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For Western analysis, striatal tissue was the same as that used for the above IP studies. Liver tissue was dounced 30 times in lysis buffer (10 mM Tris, pH 7.4, 1% Triton X-100, 150 mM NaCl, 10% glycerol, 20 mM N-ethylmaleimide, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 1 μg/mL leupeptin, 1 μg/mL aprotinin, and 20 mM NaF), allowed to rest on ice for 1 h, then centrifuged at 15,000 × g at 4 °C for 20 min and the resulting supernatant was quantitated by Lowry assay. Thirty micrograms per lane of striatal and liver soluble whole-cell extracts were run on 4 to 12% Bis-Tris MidiGels (Invitrogen) using Mops running buffer and transferred onto 0.45-μm nitrocellulose membranes. We detected transgenic human mutant HTT exon 1 protein smears in the soluble fraction by Western analysis using anti-human HTT MAB5492 (Millipore) at 1:1,000, which does not recognize endogenous mouse HTT, quantitated using Scion Image analysis software, and performed statistical analyses with GraphPad Prism 6, version 6.01. This liver tissue lysis protocol and gel system was also used to compare levels of IKKβ in oil-treated R6/1 (HD) vs. NT animals using anti-IKKβ (10AG2; Abcam) 1:500. Loading controls for striatum and liver were measured using anti–α-tubulin (Sigma-Aldrich) at 1:5,000 and anti-ERK1/2 (Cell Signaling) at 1:1,000, respectively.
Ochaba J., Fote G., Kachemov M., Thein S., Yeung S.Y., Lau A.L., Hernandez S., Lim R.G., Casale M., Neel M.J., Monuki E.S., Reidling J., Housman D.E., Thompson L.M, & Steffan J.S. (2019). IKKβ slows Huntington’s disease progression in R6/1 mice. Proceedings of the National Academy of Sciences of the United States of America, 116(22), 10952-10961.
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7

Quantitative Immunoblotting of Synaptic Proteins

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Total protein concentrations from synaptoneurosome tissue were determined using a Qubit fluorometer and Qubit protein assay kit (Invitrogen). Synaptoneurosome tissue containing approximately 15 μg total protein were heated in a sample buffer with a reducing agent (Invitrogen), loaded and then run on 8% Bis-Tris MIDI gels (Invitrogen). Tissue from each condition was loaded onto each gel. The gels were then electroblotted to a nitrocellulose membrane using an iBlot dry-blotting system (Invitrogen). Membranes were then washed in Tris-buffered saline (TBS: 150 mM NaCl/100 mM Tris base, pH 7.5) and incubated with primary antibodies diluted in blocking solution (5% Carnation nonfat dry milk in TBS-Tween) overnight at 4°C. The primary antibodies were anti-ARC (rabbit polyclonal; 1:6000, Synaptic Systems) and anti-actin (rabbit; 1:3000, Sigma). On the next day, the membranes were incubated with a secondary HRP-linked antibody (goat anti-rabbit; 1:6000, Millipore) for 1 hr. Immunoreactivity was detected using chemiluminescence (ECL Western Blot Kit; Pierce). Invitrogen markers were run on all gels to determine the relative mobility of the immunoreactive bands. For densitometric quantification of these results, the films were scanned and converted into TIF files for analysis with NIH Image J software.
McReynolds J.R., Carreira M.B, & McIntyre C.K. (2021). POST-TRAINING INTRA-BASOLATERAL COMPLEX OF THE AMYGDALA INFUSIONS OF CLENBUTEROL ENHANCE MEMORY FOR CONDITIONED PLACE PREFERENCE AND INCREASE ARC PROTEIN EXPRESSION IN DORSAL HIPPOCAMPAL SYNAPTIC FRACTIONS. Neurobiology of learning and memory, 185, 107539.
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8

Quantification of Synaptic Proteins by Western Blot

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Total protein concentrations from synaptoneurosome tissue were determined using a Qubit fluorometer and Qubit protein assay kit (Invitrogen). Synaptoneurosome tissue containing approximately 15 µg total protein were heated in a sample buffer with a reducing agent (Invitrogen), loaded and then run on 8% Bis-Tris MIDI gels (Invitrogen). Tissue from each condition was loaded onto each gel. The gels were then electroblotted to a nitrocellulose membrane using an iBlot dry-blotting system (Invitrogen). Membranes were then washed in Tris-buffered saline (TBS: 150 mM NaCl/100 mM Tris base, pH 7.5) and incubated with primary antibodies diluted in blocking solution (5% Carnation nonfat dry milk in TBS-Tween) overnight at 4°C. The primary antibodies were anti-Arc (rabbit polyclonal; 1:6000, Synaptic Systems) and anti-actin (rabbit; 1:3000, Sigma). On the next day, the membranes were incubated with a secondary HRP-linked antibody (goat anti-rabbit; 1:6000, Millipore) for 1 h. Immunoreactivity was detected using chemiluminescence (ECL Western Blot Kit; Pierce). Invitrogen markers were run on all gels to determine the relative mobility of the immunoreactive bands. For densitometric quantification of these results, the films were scanned and converted into TIF files for analysis with NIH Image J software.
McReynolds J.R., Anderson K.M., Donowho K.M, & McIntyre C.K. (2014). Noradrenergic actions in the basolateral complex of the amygdala modulate Arc expression in hippocampal synapses and consolidation of aversive and non-aversive memory. Neurobiology of learning and memory, 0, 49-57.
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9

Quantifying Synaptic Protein Levels

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Total protein concentrations from synaptoneurosome tissue were determined using a Qubit fluorometer and Qubit protein assay kit (Invitrogen). After purification, those synaptoneurosome samples containing 15 μg or more of total protein were heated in a sample buffer with a reducing agent (Invitrogen), loaded, and then run on 8% Bis-Tris MIDI gels (Invitrogen). Tissue from each condition was loaded into adjacent wells in each gel. The proteins were then transferred to a nitrocellulose membrane using an iBlot dry-blotting system (Invitrogen). Membranes were washed in Tris-buffered saline (TBS: 150 mM NaCl/100 mM Tris base, pH 7.5) and incubated with primary antibodies diluted in blocking solution (5% Carnation nonfat dry milk in TBS-Tween) overnight at 4 °C. The primary antibodies were anti-Arc (rabbit polyclonal; 1:3000, Synaptic Systems) and anti-actin (rabbit; 1:2000, Sigma–Aldrich). On the next day, the membranes were incubated with a secondary HRP-linked antibody (goat anti-rabbit; 1:6000, Millipore) for 1 h. Immunoreactivity was detected using chemiluminescence (ECL Western Blot Kit; Pierce). Invitrogen markers were run on all gels to determine the relative mobility of the immunoreactive bands. For densitometric quantification of these results, the films were scanned and converted into TIF files for analysis with NIH Image J software.
McReynolds J.R., Holloway-Erickson C.M., Parmar T.U, & McIntyre C.K. (2014). Corticosterone-induced enhancement of memory and synaptic Arc protein in the medial prefrontal cortex. Neurobiology of learning and memory, 112, 148-157.
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10

Western Blot Analysis of Protein Signaling

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Cells were harvested and lysed with 300 μL ice-cold RIPA lysis buffer (KeyGen Biotech, KGP702-100) consisting of 1 mM PMSF and protease inhibitor cocktail. The BCA Protein Assay Kit was adopted for the quantification of proteins in the lysate. Cell lysates consisting of 80 µg protein were transferred to SDS–PAGE with 8–12% gradient polyacrylamide gels (Bis-Tris Midi Gel, Life Technologies, USA), which were detected based on immunoblotting with antibodies to the relevant proteins. Similarly, β-actin antibody (Servicebio, GB13001) served as the loading control. The primary antibodies were listed as follows, including Phospho-EGF Receptor (Tyr1068, D7A5) XP® Rabbit mAb (CST, #3777), EGF Receptor (D38B1) XP Rabbit mAb (CST, #4267), Phospho-Akt (Ser473) Antibody (CST, #9271), Akt (pan) (C67E7) Rabbit mAb (CST, #4691), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb (CST, #4370), and p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb (CST, #4695). Enhanced chemiluminescence was applied for the visualization of band results following the instructions (Hercules, CA, USA).
Ding X., Ge L., Yan A., Ding Y., Tao J., Liu Q, & Qiao C. (2019). Docosahexaenoic Acid Serving As Sensitizing Agents And Gefitinib Resistance Revertants In EGFR Targeting Treatment. OncoTargets and therapy, 12, 10547-10558.
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