Mes sds running buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States

The MES SDS running buffer is a laboratory reagent used in electrophoresis techniques. It is designed to maintain the appropriate pH and ionic conditions for the separation and analysis of proteins or other macromolecules.

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NuPAGE MES SDS running buffer (157 citations) MES buffer (151 citations) MES running buffer (120 citations) Bolt MES SDS running buffer (25 citations) MES-SDS buffer (17 citations) Running Buffer (9 citations) SDS running buffer (8 citations) NuPAGE™ MES SDS Running Buffer (20X) (4 citations) MES or MOPS SDS running buffer (3 citations) MES or SDS running buffer (2 citations)

114 protocols using mes sds running buffer

Protein Quantification and Identification by Mass Spectrometry

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Protein quantification was performed using the Bradford assay according to the supplier's instructions (Bio‐Rad).
Eluates from immunoprecipitation or columns were denatured with Laemmli denaturing buffer and separated by SDS/PAGE using pre‐cast gradient gels with MOPS/SDS running buffer (NuPAGE® Novex®4–12% Bis‐Tris polyacrylamide gel, Invitrogen, Thermo Fisher Scientific). Proteins were either stained on gel using ProteoSilver Plus silver stain Kit (Sigma‐Aldrich), or electrotransferred onto a poly(vinylidene difluoride) membrane (Immobilon‐P; EMD Millipore) and immunodetected by colorimetry (alkaline phosphatase conjugated goat secondary antibodies from Promega, and NBT‐BCIP from Amresco as substrate). For identification by mass spectrometry (MS), proteins were separated by SDS/PAGE (NuPAGE® Novex®4–12% Bis/Tris polyacrylamide gel) in a MES/SDS running buffer (Invitrogen), and stained with Colloidal Blue Staining Kit (Invitrogen).

Azevedo J., Picart C., Dureau L., Pontier D., Jaquinod‐Kieffer S., Hakimi M, & Lagrange T. (2019). UAP56 associates with DRM2 and is localized to chromatin in Arabidopsis. FEBS Open Bio, 9(5), 973-985.

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Western Blot Analysis of Runx2 and Osx

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Cells were lysed using RIPA buffer (Thermo scientific, USA) containing a protease inhibitor cocktail (Roche, Switzerland). Equal amounts of protein from each sample were added to a NuPage Bis-Tris polyacrylamide gel (Invitrogen, USA) and run for 2 h using MES SDS running buffer (Invitrogen, USA). The proteins were then transferred to nitrocellulose membranes, which were blocked for 5 h at room temperature with milk (5% w/v) in Tris-buffered saline (TBS) containing Tween-20 (0.1%; TBS-T). The blots were subsequently incubated overnight with a primary antibody (1: 2,000) against Runx2 or Osx at 4 °C with oscillation, after which they were incubated with a horseradish peroxidase-conjugated secondary antibody (1: 10,000; Jackson, USA). The secondary antibodies were detected and visualized using the Super Signal West substrate (Fisher Scientific, USA). The resultant bands were quantified through densitometry with Image J software. The information of antibodies in detail showed as follows. Runx2 antibody (Abcam, ab23981, USA); Osx antibody (Abcam, ab22552, USA); Hmga2 antibody (Cell signal technology, #8179, USA).

Wang H., Sun Z., Wang Y., Hu Z., Zhou H., Zhang L., Hong B., Zhang S, & Cao X. (2016). miR-33-5p, a novel mechano-sensitive microRNA promotes osteoblast differentiation by targeting Hmga2. Scientific Reports, 6, 23170.

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SDS-PAGE Analysis of Purified armY-ACE2

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Example 12

SDS-PAGE analysis of purified armY-ACE2 was performed under non-reducing and reducing conditions and showed the expected band of ˜180 kDa (theoretical molecular weight: 150 kDa) (FIG. 13).

Briefly, 8 ul of sample buffer (Invitrogen) was added to 24 ul of eluted fractions and mixed. The sample were heated in 80° C. water bath for 10 minutes. Reducing agent (10×, Invitrogen) was added to some of the tubes containing the samples and mixed. Non-reduced and reduced samples were loaded onto a 4-12% NuPAGE pre-cast SDS-PAGE gel and separated at 175V for 30 minutes in MES-SDS running buffer (Invitrogen). PageRuler unstained protein ladder (10-200 kDa, Invitrogen) was also included. After electrophoresis, the gel was rinsed in distilled water and the protein bands stained using SimplyBlue Safe Stain (Invitrogen) and the gel photographed.

US11401309B2. Protein M fusion proteins and uses (2022-08-02). None [US]. Inventors: Jay Dela Cruz [US].

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Western Blot Analysis of Drosophila Proteins

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Fly heads were homogenized in 2× laemmli sample buffer containing 100mM DTT. Proteins were separated by SDS-PAGE at 150V for 1h using 10% Bis-Tris gels and Mes-SDS running buffer (Invitrogen). Gels were then transferred to nitrocellulose membrane, incubated in a blocking solution containing 5% milk proteins in TBST for 1h at room temperature, then probed with GFP (Cell Signaling, 2955S, 1:1000), ATG8 (custom made anti-rabbit polyclonal against peptide EP113385 (Eurogentec) [42 (link)], 1:1000,) or actin (Santa Cruz, sc-47778, 1:5,000) primary antibodies overnight at 4°C. Anti-horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam, 1:12,000) were used and blots were developed using the enhanced chemiluminescence method (ECL) according to the manufacturers’ instructions (Luminata Crescendo; Millipore). Proteins were visualized using a luminescent image analyzer (ImageQuant LAS 4000; GE Healthcare) and relative intensities measured using ImageQuant software. Proteins of interest were expressed as a ratio relative to actin levels in each sample.

Kerr F., Sofola-Adesakin O., Ivanov D.K., Gatliff J., Gomez Perez-Nievas B., Bertrand H.C., Martinez P., Callard R., Snoeren I., Cochemé H.M., Adcott J., Khericha M., Castillo-Quan J.I., Wells G., Noble W., Thornton J, & Partridge L. (2017). Direct Keap1-Nrf2 disruption as a potential therapeutic target for Alzheimer’s disease. PLoS Genetics, 13(3), e1006593.

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Extraction and Characterization of Nanoparticle Corona Proteins

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After separating and 3 times washing of hard corona SiNCs, the capsule pellet was resuspended in 100 µL of desorption buffer containing 2% SDS, 62.5 mM Tris-HCl and incubated at 95 °C for 5 min. After centrifugation at 20,000 g, 4 °C for 1 h, the supernatant containing the protein absorbed on the surface of the capsules was collected and kept at −20 °C until used. The protein concentration was determined by Pierce™ 660nm Protein Assay (Pierce, USA) according to the manufacturer’s instruction. The total amount of protein at 1.5 µg of each sample was loaded onto pre-cast Bolt™ 10% Bis-Tris Plus Gel (Invitrogen, USA) and separated in MES SDS running buffer (Invitrogen, USA) at 100 volts for 1 h 15 min. Then, the gel was stained with SilverQuest™ Silver Staining Kit (Invitrogen, USA) according to the manufacturer’s protocol.

Thiramanas R., Jiang S., Simon J., Landfester K, & Mailänder V. (2020). Silica Nanocapsules with Different Sizes and Physicochemical Properties as Suitable Nanocarriers for Uptake in T-Cells. International Journal of Nanomedicine, 15, 6069-6084.

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SDS-PAGE Analysis of Protein Samples

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Timing: ∼1–1.5 h

29.

Prepare reduced and non-reduced samples with 3 μg of protein in each sample.

30.

Reduced samples: 3 μg sample, 2.5 μL 4× Laemmli sample buffer, 3 μL 1 M DTT, and bring volume to 10 μL with gel filtration buffer.

31.

Non-reduced samples: 3 μg sample, 2.5 μL 4× Laemmli sample buffer, and bring volume to 10 μL with gel filtration buffer.

32.

Place NuPage 4%–12% Bis-Tris gel, with sticker pulled off, in the Invitrogen Mini Gel Tank, following the manufacturers detailed procedure.

33.

Using 1× NuPAGE MES SDS Running Buffer fill to the fill line of the Mini Gel Tank.a.

The total volume in the gel chamber for one side is 400 mL.

34.

Remove the comb and load 10 μL of sample in each well.

35.

Run gel at 200 V for roughly 30 min in the Invitrogen Mini Gel Tank until bands reach the bottom of the gel.

Note: Voltage may need to be lowered if running multiple gels at the same time.

36.

Stain with Coomassie blue for 20 min.

37.

De-stain with DI water until bands are prominent.

Note: We also QC our purified spike samples by negative stain EM (NSEM) analysis. The NSEM analysis method has been previously described (Edwards et al., 2021 (link)).

Stalls V., Janowska K, & Acharya P. (2022). Transient transfection and purification of SARS-CoV-2 spike protein from mammalian cells. STAR Protocols, 3(3), 101603.

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BCL2 Immunoblotting in Bcl2 Transgenic Mice

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BCL2 immunoblotting was performed using extracts from splenocytes from Bcl2^WE/WE, Bcl2^WE/+, and Bcl2^+/+ mice. Splenocytes were lysed with a lysis buffer (25 mM Hepes, 150 mM NaCl, 1% NP-40, 10 mM MgCl₂, 1 mM EDTA, 2% glycerol, and protease inhibitor cocktail tablets; Roche). Protein assays were performed with a BCA kit (Thermo Fisher Scientific). Then, 100 µg protein per sample in LDS sample buffer (Invitrogen) was run in a 4–12% Bis-Tris precast electrophoresis gel in MES SDS running buffer (Invitrogen). Proteins were then transferred onto a nitrocellulose membrane using the iBlot Dry blotting system (Thermo Fisher Scientific). Membranes were saturated by incubation for 1 h with 0.5% Tween 20, 5% milk in PBS, stained with antibodies against BCL2 (10C4; Santa Cruz Biotechnology, Inc.), and then incubated with an anti–mouse IgG-HRP Easy Blot secondary antibody (Gene Tex). Antibody binding was revealed with an ECL kit (GE Healthcare). After stripping of the membranes, staining for β-actin (AC-15; Sigma-Aldrich) was performed using the same protocol (loading control).

Viant C., Guia S., Hennessy R.J., Rautela J., Pham K., Bernat C., Goh W., Jiao Y., Delconte R., Roger M., Simon V., Souza-Fonseca-Guimaraes F., Grabow S., Belz G.T., Kile B.T., Strasser A., Gray D., Hodgkin P.D., Beutler B., Vivier E., Ugolini S, & Huntington N.D. (2017). Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival. The Journal of Experimental Medicine, 214(2), 491-510.

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Western Blot Analysis of Cav1.2 Subunit

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The cells were lysed in RIPA buffer (Thermo) containing a protease inhibitor cocktail (Roche). Equal amounts of protein from each sample were added to a NuPage Bis-Tris polyacrylamide gel (Invitrogen) and run for 2 hours using MES SDS running buffer (Invitrogen). Then, the proteins were transferred to nitrocellulose membranes and blocked for 5 hours at room temperature with milk (5% w/v) in Tris-buffered saline (TBS) with Tween-20 (0.1%; TBS-T). The blots were incubated with a primary antibody (1:200) directed against the Cav1.2 subunit overnight at 4°C with oscillation. The blots were incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000; Jackson). The secondary antibodies were detected and visualized using the Super Signal West substrate (Fisher Scientific). Densitometry measurements were made using Tanon imaging software61 (link).

Sun Z., Cao X., Zhang Z., Hu Z., Zhang L., Wang H., Zhou H., Li D., Zhang S, & Xie M. (2015). Simulated microgravity inhibits L-type calcium channel currents partially by the up-regulation of miR-103 in MC3T3-E1 osteoblasts. Scientific Reports, 5, 8077.

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Western Blot Analysis of CIRBP

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Cells were lysed with the IP lysis buffer (Pierce) as described in the protocol. Whole protein extracts (50 µg) in LDS sample buffer (Invitrogen) and DTT were applied on a NuPAGE 12% Bis-Tris-Gel (Invitrogen). The proteins were separated at constant 150 V in a MES SDS running buffer (Invitrogen). Subsequently, blotting on a PVDF membrane was performed in a full wet tank blot at 30 V. Membranes were incubated with a CIRBP rabbit polyclonal antibody recognising the C terminus of mouse CIRBP [32] (link). As secondary antibody an HRP-conjugated goat anti-rabbit (ab79051, Abcam) was used. As a loading control, an antibody to the mouse nuclear matrix protein p84 was used (ab487); with a goat to mouse HRP (ab97023) secondary antibody. The p105/p50 antibody (ab7917) was from Abcam and the IκB antibody (9242) from Cell Signaling Technology. Densitometric analysis was done using ImageJ software. The protein marker used was the prestained protein ladder V (PL005) from Geneaid.

Lopez M.A., Meier D., Wong W.W, & Fontana A. (2015). TNF induced inhibition of Cirbp expression depends on RelB NF-κB signalling pathway. Biochemistry and Biophysics Reports, 5, 22-26.

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Western Blot Analysis of Cortical Homogenate

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20μg of cortical homogenate was mixed with 4x Laemmli Sample buffer (cat # 1610747, Bio-Rad) and heated at 95 °C for 10 minutes. Samples were separated on 4-12% Bis-Tris Criterion XT Precast Gel (cat # 3450125, Bio-Rad) using MES SDS running buffer (cat # NP0002-02, Invitrogen). Protein was transferred onto 0.45μm nitrocellulose membrane (cat # 1620167, Bio-Rad) using the Trans-Blot Turbo System (Bio-Rad). Membranes were incubated in 0.1% Ponceau solution to assess transfer quality. After 3 x 5 minute 0.1% Tween-TBS buffer washes, the membrane was blocked in 5% milk for 1 hour. Primary antibodies diluted in 5 % milk were added to the membrane overnight at 4 °C (Table 1). The next day, the membrane was washed 3 x 5 minutes in 0.1% Tween-TBS buffer. Secondary antibodies diluted in 5% milk were added to the membrane for 1 hour at room temperature (Table 2). After 2 x 5 minute 0.1% Tween-TBS buffer and 1 x 5 minute TBS buffer washes, the membrane was incubated in Super Signal West Femto Chemiluminescent Substrate (cat # 34096 Thermo Scientific) for 5 minutes. Super Signal West Pico Chemiluminescent Substrate (cat # 34580 Thermo Scientific) was used for loading controls only. Membranes were imaged using the Fluor-Chem R System (Protein Simple). Densitometric analysis was performed using AlphaView Software (Protein Simple).

Di Meco A., Kemal S., Popovic J., Chandra S., Sadleir K, & Vassar R. (2022). Poloxamer-188 Exacerbates Brain Amyloidosis, Presynaptic Dystrophies, and Pathogenic Microglial Activation in 5XFAD Mice. Current Alzheimer research, 19(4), 317-329.

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