Bolt 4 to 12 bis tris gel

Manufactured by Thermo Fisher Scientific

The Bolt™ 4 to 12% Bis-Tris gels are a type of precast polyacrylamide gel used for protein electrophoresis. These gels have a gradient of 4% to 12% polyacrylamide concentration, which allows for the separation of a wide range of protein sizes. The Bis-Tris buffer system provides a neutral pH environment for protein separation.

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Lab products found in correlation

One step blue protein gel stain, by Biotium (1 mentions) Human antibody capture kit, by Cytiva (1 mentions) Hp1100 system, by Agilent Technologies (1 mentions) Biacore t200, by Cytiva (1 mentions) Series s sensor chip cm5, by Cytiva (1 mentions) Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions) Sample reducing agent, by Thermo Fisher Scientific (1 mentions) Clarity western ecl blotting substrate, by Bio-Rad (1 mentions) Bolt mops sds running buffer, by Thermo Fisher Scientific (1 mentions) Imagequant las 4000 mini, by GE Healthcare (1 mentions) Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions) Np40 cell lysis buffer, by Thermo Fisher Scientific (1 mentions) Everyblot blocking buffer, by Bio-Rad (1 mentions) Ab157107, by Abcam (1 mentions) Ab52637, by Abcam (1 mentions) Ab126613, by Abcam (1 mentions) Ab69989, by Abcam (1 mentions) Superblock blocking buffer, by Thermo Fisher Scientific (1 mentions) Odyssey infrared imaging system, by LI COR (1 mentions)

6 protocols using bolt 4 to 12 bis tris gel

Recombinant SARS-CoV-2 RBD Production

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The antigen was produced in a bioreactor based on a selected stable CHO clone. A fed-batch strategy was used for high-cell-density cultivation and expression of the RBD fusion heterodimer. Upon harvest, the cell broth was clarified by depth filtration. The clarified supernatant was further purified via sequential chromatography. The purified antigen was then buffer exchanged by tangential flow filtration and filter sterilised. Purity and integrity were evaluated by SDS-PAGE with Bolt™ 4 to 12% Bis-Tris gels (Thermo Fisher, ref. NW04120BOX), stained with One-Step Blue Protein Gel Stain (Biotium, ref. 21003), and by SEC-HPLC with an Xbridge Protein BEH SEC (Waters, ref. 186009160) connected to an HP1100 system (Agilent Technologies).
The affinity test of the RBD heterodimer with human ACE2 by surface plasmon resonance (SPR) was performed by ACROBiosystems. The Fc-tagged ACE2 (AC2-H5257, ACROBiosystems) was immobilised in a Series S Sensor Chip CM5 (Cytiva) on a Biacore T200 (Cytiva) using the Human Antibody Capture Kit (Cytiva). The affinity measure was obtained using 8 different RBD heterodimer concentrations. The antigen structure simulations were performed with UCSF ChimeraX.⁵³ (link)

Barreiro A., Prenafeta A., Bech-Sabat G., Roca M., Perozo Mur E., March R., González-González L., Madrenas L., Corominas J., Fernández A., Moros A., Cañete M., Molas M., Pentinat-Pelegrin T., Panosa C., Moreno A., Puigvert Molas E., Pol Vilarrassa E., Palmada J., Garriga C., Prat Cabañas T., Iglesias-Fernández J., Vergara-Alert J., Lorca-Oró C., Roca N., Fernández-Bastit L., Rodon J., Pérez M., Segalés J., Pradenas E., Marfil S., Trinité B., Ortiz R., Clotet B., Blanco J., Díaz Pedroza J., Ampudia Carrasco R., Rosales Salgado Y., Loubat-Casanovas J., Capdevila Larripa S., Prado J.G., Barretina J., Sisteré-Oró M., Cebollada Rica P., Meyerhans A, & Ferrer L. (2023). Preclinical evaluation of a COVID-19 vaccine candidate based on a recombinant RBD fusion heterodimer of SARS-CoV-2. iScience, 26(3), 106126.

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Western Blot Analysis of Protein Samples

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Cell lysates were prepared using NP40 Cell Lysis Buffer (Invitrogen, Carlsbad, CA, USA), supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein, determined with a Qubit™ Protein Assay Kit (Invitrogen), were prepared in 4× LDS sample buffer (Invitrogen) and 10× sample reducing agent (Invitrogen) and heated at 70 °C for 10 min. The samples were separated on Bolt 4 to 12% Bis-Tris gels in Bolt MOPS SDS running buffer (Thermo Fisher Scientific), and the proteins were transferred onto a nitrocellulose membrane (Invitrogen) using the SureLock Tandem Transfer System (Thermo Fisher Scientific). The membrane was blocked with EveryBlot Blocking Buffer (Bio-Rad), incubated with primary antibodies overnight, and incubated with secondary antibodies conjugated with horseradish peroxidase. The bands were developed using Clarity ECL Western Blotting Substrates (Bio-Rad) and visualized using an ImageQuant LAS 4000 mini (GE Healthcare, Chicago, IL, USA).

, & Chun J. (2023). Isoalantolactone Suppresses Glycolysis and Resensitizes Cisplatin-Based Chemotherapy in Cisplatin-Resistant Ovarian Cancer Cells. International Journal of Molecular Sciences, 24(15), 12397.

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Western Blot Protein Analysis Protocol

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Equal amounts of each sample (10 or 20 μg) were boiled in Laemmli sample buffer [8% glycerol, 2% SDS, 50 mM tris (pH 6.8), and 3.25% β-mercaptoethanol] for 10 min and resolved by SDS–polyacrylamide gel electrophoresis on Bolt 4 to 12% bis-tris gels (Thermo Fisher Scientific). Gels were then transferred onto nitrocellulose membranes, which were blocked in SuperBlock blocking buffer (Thermo Fisher Scientific) for 30 min at room temperature and probed with primary antibody overnight at 4°C. Membranes were then incubated with fluorophore-conjugated secondary antibodies (1:10,000) for 1 hour at room temperature. Images were captured using an Odyssey Infrared Imaging System (LI-COR Biosciences). Primary antibodies used in this study included glyceraldehyde-3-phosphate dehydrogenase (1:2000; mouse monoclonal, Abcam, ab8245), SFRP1 (1:500; rabbit monoclonal, Abcam, ab126613), MDK (1:1000: rabbit monoclonal, Abcam, ab52637), CD44 (1:1000; rabbit polyclonal, ab157107), and VGF (1:500; rabbit polyclonal, ab69989).

Higginbotham L., Ping L., Dammer E.B., Duong D.M., Zhou M., Gearing M., Hurst C., Glass J.D., Factor S.A., Johnson E.C., Hajjar I., Lah J.J., Levey A.I, & Seyfried N.T. (2020). Integrated proteomics reveals brain-based cerebrospinal fluid biomarkers in asymptomatic and symptomatic Alzheimer’s disease. Science Advances, 6(43), eaaz9360.

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Immunoblotting Analysis of METTL8 and Tagged Proteins

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To verify the loss of METTL8, cell extracts were loaded onto BOLT 4 to 12% Bis–Tris gels (ThermoFisher) followed by immunoblotting onto PVDF membrane and probing with METTL8 antibody (ThermoFisher, cat. No PA557265, 1:500 dilution) and anti-Actin C4 (EMD Millipore, cat. No MAB1501, 1:000 dilution). Expression of METTL8 in stably infected cell lines were characterized by immunoblotting with the anti-Strep antibody (THE NWSHPQFEK antibody, Genscript, cat. No. A01732, 1:1000 dilution). Transient transfections of TWIN-Strep and FLAG-tagged proteins were also loaded onto BOLT 4 to 12% Bis–Tris gels, immunoblotted to PVDF membranes, and probed with anti-TWIN-Strep (THE NWSHPQFEK antibody, Genscript, cat. No. A01732, 1:1000 dilution), anti-FLAG M2 (anti-FLAG M2, Sigma-Aldrich, 1:3000 dilution), or anti-SARS2 (Abclonal, A12297, 1:1000 dilution). Image analysis of immunoblots was performed using Image Studio software (Li-Cor).

Lentini J.M., Bargabos R., Chen C, & Fu D. (2022). Methyltransferase METTL8 is required for 3-methylcytosine modification in human mitochondrial tRNAs. The Journal of Biological Chemistry, 298(4), 101788.

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Western Blot Analysis of Protein Targets

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Cell lysates were prepared at the indicated time points postinfection in Laemmli sample buffer (Bio-Rad) supplemented with 355 mM 2-mercaptoethanol (BME). The samples were boiled for 5 min, separated on a Bolt 4 to 12% Bis-Tris gel (Invitrogen), and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% (wt/vol) nonfat dry milk in Tris-buffered saline (TBS) supplemented with 0.5% Tween 20, and proteins were detected by incubation with primary antibodies diluted in blocking buffer followed by incubation with secondary antibodies (raised in goat against the appropriate species) conjugated to horseradish peroxidase (HRP) and diluted in blocking buffer. HA was detected using an HRP-conjugated HA antibody (Roche 12013819001), V5 was detected with mouse anti-V5 tag monoclonal antibody (Invitrogen R960), SAG2A was detected using rabbit polyclonal anti-SAG2A antibodies (55 (link)), MAF1b was detected using rabbit polyclonal anti-MAF1b antibodies (27 (link)), GAPDH was detected using mouse monoclonal anti-GAPDH antibody 6C5 (Calbiochem), and biotinylated proteins were detected using streptavidin-HRP (Invitrogen S911). HRP was detected using an enhanced chemiluminescence (ECL) kit (Pierce). Silver-stained gels were generated using the Pierce silver stain kit (Thermo Scientific).

Cygan A.M., Jean Beltran P.M., Mendoza A.G., Branon T.C., Ting A.Y., Carr S.A, & Boothroyd J.C. (2021). Proximity-Labeling Reveals Novel Host and Parasite Proteins at the Toxoplasma Parasitophorous Vacuole Membrane. mBio, 12(6), e00260-21.

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Western Blotting Protocol for Protein Analysis

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Protein lysate (20 μL) was mixed with a reducing agent (Thermo Fisher) and proteins were separated using a Bolt 4% to 12% Bis-Tris Gel (Invitrogen) transferred to nitrocellulose membranes (LI-COR) by using a wet transfer system as we have described previously.¹⁹ (link) Membranes were quickly washed, blocked with LI-COR Odyssey blocking buffer and incubated with the primary antibodies overnight. The secondary antibody was incubated for 1 to 2 hours and resulting images were captured with a LI-COR imaging system. All primary antibodies and second antibodies are listed in the Supplemental Appendix.

Wang J., Tomar D., Martin T.G., Dubey S., Dubey P.K., Song J., Landesberg G., McCormick M.G., Myers V.D., Merali S., Merali C., Lemster B., McTiernan C.F., Khalili K., Madesh M., Cheung J.Y., Kirk J.A, & Feldman A.M. (2023). Bag3 Regulates Mitochondrial Function and the Inflammasome Through Canonical and Noncanonical Pathways in the Heart. JACC: Basic to Translational Science, 8(7), 820-839.

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