Bis tris page gel

Manufactured by Thermo Fisher Scientific

Bis-Tris PAGE gel is a type of polyacrylamide gel used for the separation and analysis of proteins. It is designed to maintain a neutral pH during electrophoresis, providing optimal conditions for the resolution of protein samples.

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Lab products found in correlation

A8592, by Merck Group (3 mentions) Optiprep cushion, by Merck Group (3 mentions) G8795, by Kerafast (3 mentions) Criterion tgx stain free gel, by Bio-Rad (2 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions) Sea block, by Thermo Fisher Scientific (2 mentions) Restore western blot stripping buffer, by Thermo Fisher Scientific (2 mentions) Tris glycine sds buffer, by Bio-Rad (2 mentions) Chemidoc touch system, by Bio-Rad (2 mentions) Chemidoc mp, by Bio-Rad (2 mentions) Criterion blotter system, by Bio-Rad (2 mentions) Trans blot turbo system, by Bio-Rad (2 mentions) Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions) Biospectrum imaging system, by Analytik Jena (2 mentions) Clarity western ecl blotting substrate, by Bio-Rad (2 mentions) Gapdh, by Merck Group (2 mentions) Vsv m, by Kerafast (2 mentions) Supersignal west pico chemiluminescent substrate kit, by Thermo Fisher Scientific (2 mentions) Pai 7218, by Thermo Fisher Scientific (2 mentions) Ab9044, by Abcam (2 mentions)

Close spelling variants of this product

Bis-Tris gels (1248 citations) SDS PAGE 4–12% Bis-Tris gels (7 citations) Bolt SDS-PAGE 4%–12% Bis-Tris gels (6 citations) 4-to-12% bis-Tris SDS-PAGE gels (4 citations) 4–16% Native PAGE Bis-Tris gels (4 citations) NuPAGE Bis-Tris gradient SDS-PAGE gels (3 citations) Nu‐PAGE Novex 10% Bis‐Tris gels (3 citations) PAGE gels (2 citations) NuPAGE® Novex 4–12% (w/v) Bis-Tris SDS-PAGE gels (2 citations) SDS–PAGE 4–12% Bis–Tris Protein Gels (2 citations)

14 protocols using bis tris page gel

Western Blot Analysis of Shh-Signaling Proteins

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Lysates from 3T3-[Shh-BlastR;Cas9] cells were prepared in SDS sample buffer (50 mM Tris HCl pH 6.8, 8% v/v glycerol, 2% w/v SDS, 100 mM DTT, 0.1 mg/mL bromophenol blue), boiled and sonicated. Samples were loaded onto a 4–15% Criterion TGX Stainfree gel (Bio-Rad), and run for 25 min, 300V in Tris/Glycine/SDS buffer (Bio-Rad), before being transferred onto a PVDF membrane using a Transblot Turbo system (Bio-Rad). Membranes were blocked in 1:1 PBS:SeaBlock (Thermo Scientific) for 1 h at room temperature, and subsequently incubated with the indicated primary antibody for 16 h at 4 °C (Supplementary Table 10). After incubation with HRP-conjugated secondary antibody, blots were developed using Supersignal West Femto Maximum Sensitivity Substrate (Thermo Fisher) and imaged on a ChemiDoc MP (Bio-Rad). Membranes were stripped using Restore Western Blot stripping buffer (Thermo-Fisher) and re-probed as described.
For analysis of immunoprecipitations, Western blotting was performed as described above, except samples were separated in 4–12% Bis-Tris PAGE gels (Invitrogen) using MOPS running buffer, transferred to PVDF membranes using the Criterion Blotter system (Bio-Rad), developed using ECL or ECL 2 chemiluminescence detection kits (Pierce), and imaged on a Chemidoc Touch system (Bio-Rad).

Breslow D.K., Hoogendoorn S., Kopp A.R., Morgens D.W., Vu B.K., Kennedy M.C., Han K., Li A., Hess G.T., Bassik M.C., Chen J.K, & Nachury M.V. (2018). A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies. Nature genetics, 50(3), 460-471.

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Western Blot Analysis of Shh-Signaling Proteins

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Quantifying Nascent Protein Translation

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We analyzed nascent translation using two methods: ³⁵S and ribopuromycilation. To conduct ³⁵S metabolic labeling, cells were incubated in methionine-free RPMI (Gibco) containing 11 μCi EasyTag EXRESS35S Protein Labeling mix (Perkin Elmer) for 15 minutes at 37 °C. Cells were directly lysed in NuPage LDS sample buffer. Lysates were boiled at 95 °C for 10 minutes, then separated on 10% BisTris PAGE gels (Invitrogen). Gels were stained with coomassie for total protein visualization, then analyzed by phosphorimaging (Typhoon FLA 7000, GE). For ribopuromycilation assay, cells were treated with 10 μg/ml puromycin (Invitrogen) in culture medium for 5 minutes at 37 °C. Cells were lysed and separated by PAGE as above. For western blotting proteins were transferred to PVDF membranes (Life Technologies) and stained with Ponceau Red to visualize total proteins. The membrane was washed and blocked in 5% non-fat dry milk in TBST. Membranes were probed with 1:1000 dilution of mouse anti-puromycin antibody (EMD Millipore), followed by 1:10,000 dilution of horseradish peroxidase-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch).

Rath S., Prangley E., Donovan J., Demarest K., Wingreen N.S., Meir Y, & Korennykh A. (2019). Concerted 2-5A-Mediated mRNA Decay and Transcription Reprogram Protein Synthesis in dsRNA Response. Molecular cell, 75(6), 1218-1228.e6.

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Streptavidin-Based Biotinylated Protein Elution

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Biotinylated proteins were eluted from streptavidin beads by the addition of 20 μL elution buffer (2X Laemmli sample buffer, 20 mM DTT, 2 mM biotin) followed by a 10 min incubation at 95°C. Proteins were resolved by 4%–12% Bis-Tris PAGE gels (Thermo Fisher) and transferred to nitrocellulose membrane (Thermo Fisher). After blocking with 3% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween 20 (TBST; Thermo Fisher) for 1 hour, membrane was incubated with 0.3 mg/mL HRP-conjugated streptavidin for one hour. The Clarity Western ECL blotting substrate (Bio-Rad) and BioSpectrum imaging system (UVP) were used to develop and detect chemiluminescence.

Xie Q., Li J., Li H., Udeshi N.D., Svinkina T., Orlin D., Kohani S., Guajardo R., Mani D., Xu C., Li T., Han S., Wei W., Shuster S.A., Luginbuhl D.J., Quake S.R., Murthy S.E., Ting A.Y., Carr S.A, & Luo L. (2022). Transcription Factor Acj6 Controls Dendrite Targeting via A Combinatorial Cell-Surface Code. Neuron, 110(14), 2299-2314.e8.

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Photoconjugation of PpL Mutants and Antibodies

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In initial screens, PpL mutants and anti-CD3 (clone UTH1, BD bioscience 555329) antibodies were diluted into PBS pH7.6 such that the final concentrations were approximately 50 µM and 2 µM, respectively, and loaded into thin walled 200 µL polypropylene microtubes (PCR tubes). This mixture was then irradiated for 1 h under 365 nm light at an intensity of 6.4 mW/cm² from an LED source (M365LP1, Thor Labs) 14 cm away. Products were reduced using DTT solution (ThermoFisher) and separated on 4–12% BisTris PAGE gels (ThermoFisher) to observe photoconjugation. Full gel images are shown in Supplementary Fig. S2. Photocleavage was accomplished using the same irradiation setup. Photoconjugations to anti-FLAG antibody (anti-DYDDDDK clone 1557CT661.18.1, Lifespan Biosience LS‑C392574) or Cetuximab (Selleck Chemicals A2000) were done identically, with 100 µM PpL constructs that had been freshly purified. Photoconjugates were then purified from excess PpL using Amicon Ultra 50 kDa MWCO spin filter columns (Millipore-Sigma, UCFC505008). The unaltered antibody control conditions of our ELISA experiments, described below, validated Anti-FLAG binding to the FLAG peptide and Cetuximab binding to EGFR protein.

Brasino M., Wagnell E., Hamilton S., Ranganathan S., Gomes M.M., Branchaud B., Messmer B, & Ibsen S.D. (2022). Turning antibodies off and on again using a covalently tethered blocking peptide. Communications Biology, 5, 1357.

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Biotinylated Protein Detection via Streptavidin

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Biotinylated proteins were eluted from streptavidin beads by the addition of 20 μL elution buffer (2X Laemmli sample buffer, 20 mM DTT, 2 mM biotin) followed by a 10 min incubation at 95°C. Proteins were resolved by 4%-12% Bis-Tris PAGE gels (Thermo Fisher) and transferred to nitrocellulose membrane (Thermo Fisher). After blocking with 3% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween 20 (TBST; Thermo Fisher) for 1 hour, membrane was incubated with 0.3 mg/mL HRP-conjugated streptavidin for one hour. The Clarity Western ECL blotting substrate (Bio-Rad) and BioSpectrum imaging system (UVP) were used to develop and detect chemiluminescence.

Luo L., Xie Q., Li J., Li H., Udeshi N.D., Svinkina T., Kohani S., Guajardo R., Mani D., Xu C., Li T., Han S., Wei W., Shuster S.A., Luginbuhl D.J., Ting A.Y., & Carr S.A. (2021). Transcription Factor Control of Dendrite Targeting via Combinatorial Cell-Surface Codes.

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Purification and Analysis of SARS-CoV-2 Spike Pseudotypes

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293T cells transfected with CoV spike pseudotypes (producer cells) were lysed in 1% sodium dodecyl sulfate, 150mM NaCl, 50 mM Tris-HCl and 5 mM EDTA and centrifuged at 14,000g for 20 minutes. Pseudotyped particles were concentrated from producer cell supernatants that were overlaid on a 10% OptiPrep cushion in PBS (Sigma–Aldrich) and centrifuged at 20,000g for 2h at 4 °C. Lysates and concentrated particles were analyzed for FLAG (Sigma–Aldrich; A8592; 1:10,000), GAPDH (Sigma–Aldrich; G8795; 1:10,000) and/or VSV-M (Kerafast; 23H12; 1:5,000) expression on 10% Bis-Tris PAGE gel (Thermo Fisher Scientific).

Wells H.L., Letko M., Lasso G., Ssebide B., Nziza J., Byarugaba D.K., Navarrete-Macias I., Liang E., Cranfield M., Han B.A., Tingley M.W., Diuk-Wasser M., Goldstein T., Johnson C.K., Mazet J., Chandran K., Munster V.J., Gilardi K, & Anthony S.J. (2021). The evolutionary history of ACE2 usage within the coronavirus subgenus Sarbecovirus. bioRxiv.

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Pseudotyped Particle Production and Analysis

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Producer cells (spike-transfected 293T) were lysed in 1% sodium dodecyl sulfate, 150 mM NaCl, 50 mM Tris-HCl and 5 mM EDTA and clarified by centrifugation at 14,000g for 20 min.
Pseudotyped particles were concentrated from producer cell supernatants that were overlaid on a 10% OptiPrep cushion in PBS (Sigma–Aldrich) and centrifuged at 20,000g for 2 h at 4 °C. Lysates and concentrated particles were analysed for FLAG (Sigma–Aldrich; A8592; 1:10,000), GAPDH (Sigma–Aldrich; G8795; 1:10,000) and/or VSV-M (Kerafast; 23H12; 1:5,000) expression on 10% Bis-Tris PAGE gel (Thermo Fisher Scientific).

Letko M., Marzi A, & Munster V. (2020). Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nature Microbiology, 5(4), 562-569.

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Purification and Analysis of Pseudotyped Viral Particles

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293T cells transfected with CoV spike pseudotypes (producer cells) were lysed in 1% sodium dodecyl sulfate, 150 mM NaCl, 50 mM Tris-HCl, and 5 mM EDTA and centrifuged at 14,000g for 20 minutes. Pseudotyped particles were concentrated from producer cell supernatants that were overlaid on a 10% OptiPrep cushion in PBS (Sigma–Aldrich) and centrifuged at 20,000g for 2 h at 4°C. Lysates and concentrated particles were analyzed for FLAG (Sigma–Aldrich; A8592; 1:10,000), GAPDH (Sigma–Aldrich; G8795; 1:10,000), and/or VSV-M (Kerafast; 23H12; 1:5,000) expression on 10% Bis-Tris PAGE gel (Thermo Fisher Scientific).

Wells H.L., Letko M., Lasso G., Ssebide B., Nziza J., Byarugaba D.K., Navarrete-Macias I., Liang E., Cranfield M., Han B.A., Tingley M.W., Diuk-Wasser M., Goldstein T., Johnson C.K., Mazet J.A., Chandran K., Munster V.J., Gilardi K, & Anthony S.J. (2021). The evolutionary history of ACE2 usage within the coronavirus subgenus Sarbecovirus. Virus Evolution, 7(1), veab007.

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Purification of GFP-tagged Protein

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Co-transform bacteria with sfGFP-151-quad and qtRNA expression plasmids. Inoculate a single colony of the expression strain in DRM (with 100 µg/mL spectinomycin, 30 µg/mL kanamycin, and 25 mM glucose) as the seed culture; grow overnight at 37°C, 200 rpm. Dilute 1:1000 and into DRM that induces qtRNA expression (with 100 µg/mL spectinomycin, 30 µg/mL kanamycin, and 1 mM IPTG) and grow for 29 hr, 200 rpm, at 37°C. Cultures were spun down at 5000 g for 10 min and the pellet frozen at –80°C. Thawed pellets were lysed using B-PER Complete Reagent (Thermofisher 89821) and His-tagged protein was purified from cell lysate using a Ni-NTA spin column (Qiagen, 31014). The resulting product was run on a 12% Bis-Tris PAGE gel (Thermofisher, NP0342PK2), and the appropriate band was extracted for mass spectrometry analysis.

DeBenedictis E.A., Söll D, & Esvelt K.M. (2022). Measuring the tolerance of the genetic code to altered codon size. eLife, 11, e76941.

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