Mes running buffer

Manufactured by Bio-Rad

Sourced in United States

MES running buffer is a laboratory reagent used in electrophoresis applications. It is designed to maintain the appropriate pH and ionic conditions for the separation and analysis of biomolecules, such as proteins and nucleic acids.

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Lab products found in correlation

Criterion xt, by Bio-Rad (5 mentions) Proteinase k, by Merck Group (2 mentions) Coommassie brilliant blue, by Bio-Rad (2 mentions) Coomassie brilliant blue, by Bio-Rad (2 mentions) Horseradish peroxidase conjugate secondary antibody, by Bio-Rad (1 mentions) 2 deoxy d glucose, by Merck Group (1 mentions) Dorsomorphin, by Abcam (1 mentions) Bca protein assay, by Thermo Fisher Scientific (1 mentions) Antibodies against gapdh sc 25778, by Santa Cruz Biotechnology (1 mentions) Cst2532, by Cell Signaling Technology (1 mentions) Polyvinylidene fluoride pvdf immobilon p membrane ipvh00010, by Merck Group (1 mentions) Protein molecular weight marker rpn800e, by GE Healthcare (1 mentions) Compound c, by Abcam (1 mentions) A769662, by Abcam (1 mentions) Histone h1, by Merck Group (1 mentions) Criterion xt bis tris gel, by Bio-Rad (1 mentions) Matrigel, by Merck Group (1 mentions) Bis trisacrylamide gels, by Bio-Rad (1 mentions) Anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions) Odyssey clx imager, by LI COR (1 mentions)

Close spelling variants of this product

XT MES running buffer (15 citations) Running buffer (12 citations) MES buffer (3 citations) 1X2-(N-morpholino)ethanesulfonic acid (MES) running buffer (2 citations) 2-morpholinoethanesulfonic acid (MES) running buffer (2 citations) XT MES Running Buffer 20X (2 citations)

10 protocols using mes running buffer

AMPK Signaling Pathway Analysis

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Antibodies against GAPDH (sc-25778) were purchased from Santa Cruz Biotechnology. Antibodies against phospho-ACC (Ser⁷⁹) (CST3661), phospho-AMPKα (Thr¹⁷²) (CST2535) and total AMPK (CST2532) were purchased from Cell Signaling Technology. Bis-Tris 4–12% polyacrylamide gels (345–0123), MES running buffer (161–0789) and horseradish peroxidase secondary antibody conjugate (170–6515) were obtained from Bio-Rad. Polyvinylidene Fluoride (PVDF) Immobilon-P membrane (IPVH00010) was manufactured by Merck Millipore. Protein molecular weight marker (RPN800E) was purchased from GE Healthcare Life Sciences, while enhanced chemiluminescence (ECL) reagent (32209) and BCA protein assay were purchased from Life Technologies. Metformin was obtained from Calbiochem (Merck Millipore), 2-deoxy-D-glucose from Sigma-Aldrich and Compound C (Dorsomorphin) and A-769662 from Abcam. All other reagents, unless otherwise specified, were purchased from Sigma-Aldrich or Merck Millipore.

Bizjak M., Malavašič P., Dolinar K., Pohar J., Pirkmajer S, & Pavlin M. (2017). Combined treatment with Metformin and 2-deoxy glucose induces detachment of viable MDA-MB-231 breast cancer cells in vitro. Scientific Reports, 7, 1761.

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Cdk1-cyclin B1 Kinase Assay

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The activity of recombinant Cdk1-cyclin B1 complex was measured with histone H1 as a model substrate as described elsewhere (Murray, 1991 ). In brief, different concentrations of Cks2 protein (0–10 μM) and histone H1 (0.5 mg/mL, Millipore) were added into kinase assay buffer (5 mM Tris pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mg/mL ovalbumin, 1 μM okadaic acid, 100 μM ATP) plus γ-³²P-ATP. Protein samples were resolved in a Bis-Tris gel with MES running buffer (Bio-Rad) and transferred to a PVDF membrane. Phosphorylation of histone H1 was quantified by autoradiography.

Ha S.H., Kim S.Y, & Ferrell JE J.r. (2016). The prozone effect accounts for the paradoxical function of the Cdk-binding protein Suc1/Cks. Cell reports, 14(6), 1408-1421.

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SDS-PAGE Fractionation and Mass Spectrometry

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Volumes of 15 µg protein equivalent were diluted with reducing LDS (lithium dodecyl sulfate) sample buffer (5% β-mercaptoethanol) for SDS-PAGE, heated 5 min at 85 °C, loaded on an 18-well Criterion XT BisTris gel, and electrophoresed in MES running buffer (Bio-Rad, CA, USA). After fixation and staining with BioSafe stain (Bio-Rad), the sample lanes were divided into 6 equal vertical segments to fractionate the proteins by size prior to nano-flow liquid chromatography–electrospray tandem mass spectrometry (nanoLC–ESI–MS/MS) analysis. Segments were cut horizontally across all lanes using major protein bands common to all samples as guides. Each gel piece was cut into ~2mm pieces, transferred to PCR tubes, and stored in 200 mM Tris at 4 °C until trypsin digest.

Cabello-Arreola A., Ho A.M., Ozerdem A., Cuellar-Barboza A.B., Kucuker M.U., Heppelmann C.J., Charlesworth M.C., Ceylan D., Stockmeier C.A., Rajkowska G., Frye M.A., Choi D.S, & Veldic M. (2020). Differential Dorsolateral Prefrontal Cortex Proteomic Profiles of Suicide Victims with Mood Disorders. Genes, 11(3), 256.

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Proteinase K Digestion of Fibrils

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The fibrils used for the ProK digestion were prepared as above and not subjected to sonication. Briefly, 10 μg of fibrils was taken in 20 μl of 1× PBS, added with increasing concentrations (0.0, 0.5, 1.0, 1.5, and 2.0 μg/ml) of ProK and incubated for 37°C for 30 min. Following incubation, the reaction was quenched by directly adding 5 μl of 5× Laemmli buffer to the reaction mixture and boiling the samples for 8 min at 95°C. The digested fibrillar products were separated by SDS-PAGE using precast 12% bis-tris gels (Bio-Rad, Criterion XT) with MES running buffer (Bio-Rad). Bands were visualized using the Coomassie blue staining.

Kumar S.T., Mahul-Mellier A.L., Hegde R.N., Rivière G., Moons R., Ibáñez de Opakua A., Magalhães P., Rostami I., Donzelli S., Sobott F., Zweckstetter M, & Lashuel H.A. (2022). A NAC domain mutation (E83Q) unlocks the pathogenicity of human alpha-synuclein and recapitulates its pathological diversity. Science Advances, 8(17), eabn0044.

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Proteinase K-Mediated Protein Digestion

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Ten micrograms of protein from aggregation reactions were incubated with Proteinase K (Sigma Aldrich P2308) at the indicated concentrations (Figures 2d, 3c, 3d) for 30 min at 37 °C. Reactions were quenched by the addition of sample loading buffer (2% final SDS concentration) and boiling at 95 °C for 10 min. Digestion products were separated by SDS-PAGE using precast 12% Bis-Tris gels (Bio-Rad, Criterion XT) with MES running buffer (Bio-Rad). Bands were visualized with Coommassie Brilliant Blue (Bio-Rad).

Balana A.T., Levine P.M., Craven T.W., Mukherjee S., Pedowitz N.J., Moon S.P., Takahashi T.T., Becker C.F., Baker D, & Pratt M.R. (2021). O-GlcNAc modification of small heat shock proteins enhances their anti-amyloid chaperone activity. Nature chemistry, 13(5), 441-450.

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Proteinase K-Mediated Protein Digestion

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Proteinase K Digestion of α-synuclein Aggregates

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Unmodified α-synuclein aggregates (35 μM), ubiquitin-thiol (35 μM), or both were incubated in 20 μL of 1 DPBS with 5 mM CaCl₂ and 0, 1, and 2 μg m L ⁻¹ proteinase K for 30 min at 37 °C. SDS loading buffer was added (2% final SDS concentration), and samples were boiled for 10 min at 95 °C to halt the digestion. Products were analyzed by SDS-PAGE using precast 10% Bis-Tris gels (Bio-Rad, Criterion XT) with MES running buffer (Bio-Rad) and stained with Coomassie Brilliant Blue (Bio-Rad).

Moon S.P., Balana A.T., Galesic A., Rakshit A, & Pratt M.R. (2019). Ubiquitination Can Change the Structure of the α-Synuclein Amyloid Fiber in a Site Selective Fashion. The Journal of organic chemistry, 85(3), 1548-1555.

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Transfection and Membrane Repair Analysis of Nanodysferlins

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Transfection of HEK293 and COS7 cells was as described.³⁷ (link) Expression of the nanodysferlins as Venus fusion proteins was assayed by SDS-PAGE, transfer to PVDF membrane, and immunoblotting with Hamlet anti-dysferlin, as reported.³¹ (link) Prior to electrophoresis, cell pellets were extracted with RIPA buffer. The extracts were incubated with three volumes of 2× concentrated SDS-PAGE sample buffer for 15 min at 37°C and analyzed in 4%–12% Bis-Tris acrylamide gels in MES running buffer (all reagents from BioRad, Hercules, CA, USA).
FDB muscles were transfected by electroporation, as described.³⁰ (link)^,³¹ (link)^,⁶⁸ ^,⁶⁹ Muscles were removed 1–2 weeks after electroporation and either dissociated and cultured on a layer of Matrigel (Sigma-Aldrich, St. Louis, MO, USA) to study Ca²⁺ signaling and t-tubule localization, or teased into small bundles to assess membrane repair.

Muriel J., Lukyanenko V., Kwiatkowski T.A., Li Y., Bhattacharya S., Banford K.K., Garman D., Bulgart H.R., Sutton R.B., Weisleder N, & Bloch R.J. (2024). Nanodysferlins support membrane repair and binding to TRIM72/MG53 but do not localize to t-tubules or stabilize Ca2+ signaling. Molecular Therapy. Methods & Clinical Development, 32(2), 101257.

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Western Blot Protein Analysis Protocol

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Samples were analyzed by western blotting as previously described^{12 (link)}. Briefly, clarified cell lysates were boiled in LDS sample buffer (Life Technologies) at 95 °C for five minutes, and resolved by electrophoresis on 4–12% gels (Bio-Rad) using MES running buffer (Bio-Rad). Proteins were transferred to nitrocellulose membranes (Life Technologies) using an iBlot® device (Life Technologies) and membranes were blocked in blocking buffer (LI-COR Biosciences) for 1 hour at room temperature. Membranes were probed with primary antibodies (Cell Signaling Technology) in 5% BSA (Sigma), 0.1% Tween-20 tris-buffered saline solution (TBS-T) overnight at 4–8 degrees Celsius, washed 3 times for 10 minutes in TBS-T, followed by incubation with an anti-rabbit secondary antibody (Licor) in 5% milk (Cell Signaling Technologies) TBS-T for 45 minutes. After 3 additional 5-minute washes in TBS-T, bands were visualized on a LI-COR ODYSSEY® CLx imager. Protein bands were quantified using Image Studio (version 3.1.4) software.

Camblin A.J., Tan G., Curley M.D., Yannatos I., Iadevaia S., Rimkunas V., Mino-Kenudson M., Bloom T., Schoeberl B., Drummond D.C., Lugovskoy A.A., Louis C.U, & Askoxylakis V. (2019). Dual targeting of IGF-1R and ErbB3 as a potential therapeutic regimen for ovarian cancer. Scientific Reports, 9, 16832.

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Proteinase K Digestion of Ubiquitin-Modified α-Synuclein

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Ubiquitin-modified α-synuclein aggregates (10 μg) were incubated in 20 μL of 1× DPBS, 5 mM CaCl₂, 5 mM DTT, and 0, 1, and 2 μg mL⁻¹ proteinase K for 30 min at 37 °C. SDS loading buffer was added (2% final SDS concentration), and samples were boiled for 10 min at 95 °C. Products were analyzed by SDS-PAGE using precast 10% Bis-Tris gels (Bio-Rad, Criterion XT) with MES running buffer (Bio-Rad) and stained with Coomassie Brilliant Blue (Bio-Rad).

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