Tris hcl ph 8

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

Tris-HCl pH 8.0 is a buffer solution commonly used in various laboratory applications. It consists of a mixture of tris(hydroxymethyl)aminomethane (Tris) and hydrochloric acid (HCl), providing a stable pH of 8.0.

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Lab products found in correlation

Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (4 mentions) Sodium chloride (nacl), by Merck Group (4 mentions) Glutathione reduced, by GoldBio (3 mentions) L arginine, by Merck Group (3 mentions) Ez link nhs peg4 biotin, by Thermo Fisher Scientific (2 mentions) Ultrapure water, by Thermo Fisher Scientific (2 mentions) Sorbitan monooleate span 80, by Tokyo Chemical Industry (2 mentions) Silicone coated glasses, by Matsunami (2 mentions) Ds 11 fx, by DeNovix (2 mentions) Dithiothreitol (dtt), by Thermo Fisher Scientific (2 mentions) Polyethylenimine transfection, by Polysciences (2 mentions) Laemmli dye, by Merck Group (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Edta ph 8, by Thermo Fisher Scientific (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions) Bioruptor pico, by Diagenode (2 mentions) Acetonitrile, by Thermo Fisher Scientific (2 mentions) Sypro orange dye, by Thermo Fisher Scientific (2 mentions) Prism 9, by GraphPad (2 mentions) Glutathione (gsh), by GoldBio (2 mentions)

Close spelling variants of this product

Tris-HCl pH Tris-HCl

23 protocols using tris hcl ph 8

Cross-linking and Chromatin Preparation

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10 million cells were cross-linked in medium with 1% formaldehyde for 8 min at RT on a slow shaker, quenched with freshly prepared 0.125M glycine, incubated 5min at RT on a slow shaker, then pelleted at 400 g for 5 minutes at 4°C, washed three times with 30ml of ice cold PBS and then incubated for 20 minutes rotating at 4°C in 1mL of RIPA lysis buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0 (Invitrogen), 140mM NaCl, 1% (v/v) Triton X-100 (Euromedex), 0.1% (v/v) SDS and 0.1% sodium deoxycholate (SIGMA)). Nuclei were pelleted at 1350 g for 5 minutes at 4°C, washed for 10 minutes rotating with 1ml of a buffer containing 10mM Tris, 200mM NaCl, 1mM EDTA (Invitrogen), 0.5 mM EGTA (Euromedex), pelleted and lysed in buffer containing 0.4% SDS (Euromedex), 10mM EDTA (Invitrogen), 50mM Tris-HCl pH 8.0 for 30min on ice (volume of buffer = 100μl/1.6 million cells). Lysates were sonicated on a Bioruptor Pico (Diagenode) sonication devices (11cycles 30 s ON, 30 s OFF) to reach fragments ranging from 150 to 500bp, and then centrifuged at 10,000 g for 10 minutes at 4°C to remove debris. Samples were then snap-frozen in liquid nitrogen and stored at −80°C until immunoprecipitation. All buffers contained cOmplete EDTA free Protease inhibitor cocktail (Roche).

Gentili M., Lahaye X., Nadalin F., Nader G.F., Puig Lombardi E., Herve S., De Silva N.S., Rookhuizen D.C., Zueva E., Goudot C., Maurin M., Bochnakian A., Amigorena S., Piel M., Fachinetti D., Londoño-Vallejo A, & Manel N. (2019). The N-Terminal Domain of cGAS Determines Preferential Association with Centromeric DNA and Innate Immune Activation in the Nucleus. Cell Reports, 26(9), 2377-2393.e13.

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HLA-B*18:01 Heavy Chain Refolding

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The sequence encoding HLA‐B*18:01 heavy chain was obtained from the IPD‐IMGT/HLA database.
¹⁴ (link) The cDNA construct for the soluble protein insert, excluding the transmembrane domain, was sub‐cloned into the pET30 plasmid vector using NedI/HindIII restriction enzymes (GenScript, Piscataway, USA). The recombinant protein and human β2‐microglobulin (β2m) were then individually expressed in BL21‐RIL Escherichia coli cells, resulting in inclusion bodies that were extracted and purified through centrifugation.
⁴⁹ (link) The peptides described in this study were chemically synthesised (GenScript; Tables 1 and 2). Refolding was performed using 5 mg of peptide, 30 mg of HLA‐B*18:01 heavy chain and 10 mg of β2m in refolding buffer [3 m Urea (ThermoFisher, Scoresby, VIC, Australia), 0.4 m l‐Arginine, (Merck, Darmstadt, Germany) 0.1 m Tris–HCl pH 8 (ThermoFisher), 2 mm Na‐EDTA pH 8 (Merck), 0.16% w/v reduced Glutathione (Goldbio, St Louis, USA) and 0.03% w/v oxidised Glutathione (Goldbio)]. This solution was dialyzed in 10 mM Tris–HCl pH 8 (ThermoFisher) and the peptide‐HLA‐B*18:01 complexes were purified using a two‐stage anion exchange chromatography (Cytiva, Marlborough, Massachusetts, USA).

Leong S.L., Murdolo L., Maddumage J.C., Koutsakos M., Kedzierska K., Purcell A.W., Gras S, & Grant E.J. (2024). Characterisation of novel influenza‐derived HLA‐B*18:01‐restricted epitopes. Clinical & Translational Immunology, 13(5), e1509.

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Recombinant pHLA Complex Production

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DNA plasmids encoding HLA‐A*03:01 α‐chain and β2‐microglobulin were transformed separately into a BL21 strain of Escherichia Coli, as previously described.⁶³ Recombinant proteins were expressed individually, where inclusion bodies were extracted and purified from the transformed E. coli cells. Soluble pHLA complexes were produced by refolding 30 mg of HLA‐A*03:01 with 10 mg of β‐2‐microglobulin and 5 mg of either NP_265‐IAV, NP_323‐IBV or NP_270‐ICV peptide (Genscript, Piscataway, USA) into a buffer of 3 m Urea (Univar solutions, USA), 0.5 m L‐Arginine (Sigma‐Aldrich), 0.1 m Tris–HCl pH 8.0 (Fisher Bioreagents), 2.5 mm EDTA pH 8.0 (Sigma‐Aldrich, St Louis, USA), 5 mm Glutathione (reduced) (Goldbio, St Louis, USA) and 1.25 mm Glutathione (oxidised; Goldbio) for 3 h. The refold mixture was dialysed into 10 mm Tris–HCl pH 8.0 (Fisher Bioreagents), and soluble pHLA was purified using anion exchange chromatography using a HiTrapQ column (GE Healthcare).

Nguyen A.T., Lau H.M., Sloane H., Jayasinghe D., Mifsud N.A., Chatzileontiadou D.S., Grant E.J., Szeto C, & Gras S. (2022). Homologous peptides derived from influenza A, B and C viruses induce variable CD8 + T cell responses with cross‐reactive potential. Clinical & Translational Immunology, 11(10), e1422.

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Serum Biotin Conjugation Protocol

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Sample IDs were recoded and all samples were randomized before biotinylation. Biotinylation steps were performed on ice or at 4°C in a Nunc™ 96-well polypropylene plate (Thermo Fisher Scientific, Waltham, USA). Briefly, crude serum samples were centrifuged at 16 000 × g, and 10 μl cleared serum was diluted in PBS (D-PBS-Sterile w/o Mg, Ca, GE Healthcare Life Sciences, Marlborough, USA) after which 2.56 mM Biotin (EZ-LinkTM NHS-PEG4-Biotin, Thermo Fisher Scientific) solution diluted in PBS (GE Healthcare Life Sciences) was added, to a final biotin concentration of 1.13 mM. The reaction was terminated after 2 hours by adding 0.5 M Tris-HCl pH 8.0 (Thermo Fisher Scientific) to a final concentration of 181 mM. For each biotinylation plate, three replicates of a reference serum sample (ERM-DA470k/IFCC) ^{[15 (link)]} were included as process control. Biotinylated samples were aliquoted and stored at −80°C.

Emruli V.K., Liljedahl L., Axelsson U., Richter C., Theorin L., Bjartell A., Lilja H., Donovan J., Neal D., Hamdy F.C, & Borrebaeck C.A. (2021). Identification of a serum biomarker signature associated with metastatic prostate cancer. Proteomics. Clinical applications, 15(2-3), e2000025.

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Biotin-Affinity Purification of Protein Complexes

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Cells were lysed on ice in 550 µl (six-well plates) or 1 ml (10 cm dishes) of RIPA buffer (Boston Bioproducts) in presence of cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail (Millipore Sigma) and PhosSTOP (Millipore Sigma) for 10 min. Lysates were cleared by centrifugation at 16,000 × g for 10 min at 4 °C. 10% of the lysed cells was set aside as input. set aside as input. Pull down and washes were performed as in ref. ^{27 (link)}. Briefly, lysates were mixed with Pierce Streptavidin Magnetic Beads (Thermo Fisher) at a ratio of 100 µl beads/4 million cells. Lysates were incubated with beads with constant rotation for one hour at room temperature and then overnight (O/N) at 4 °C. Beads were then applied to a magnet and subjected to the following washes: two times with 1 ml of RIPA, one time with 1 M 1 ml of KCl (Thermo Fisher), one time with 1 ml of 0.1 M Na₂CO₃ (VWR), one time with 1 ml of freshly prepared 2 M Urea (VWR) resuspended in 10 mM Tris-HCl pH 8.0 (Thermo Fisher) and two times with RIPA. Proteins were eluted from beads by adding 150 µl (six-well plates) or 500 µl (10 cm dish) of non-reducing Laemmli (Boston bioproducts) containing 20 mM DTT (Thermo Fisher) and 2 mM biotin (Cayman chemicals) and boiled for 20 min. Input was diluted with 2X sample buffer (Sigma). For mass spectrometry, beads were processed as follows.

Gentili M., Liu B., Papanastasiou M., Dele-Oni D., Schwartz M.A., Carlson R.J., Al’Khafaji A.M., Krug K., Brown A., Doench J.G., Carr S.A, & Hacohen N. (2023). ESCRT-dependent STING degradation inhibits steady-state and cGAMP-induced signalling. Nature Communications, 14, 611.

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Synthetic Lipid Nanoparticle Preparation

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DNAs were purchased from
Eurofins Genomics
(Tokyo, Japan). Nonmodified and FAM-modified DNAs were of oligonucleotide
purification cartridge grade, and Alexa 405- and Cy3-modified DNAs
were of high-performance liquid chromatography purification grade.
DNA was stored in Ultrapure water (18 MΩ·cm in resistance)
at a concentration of 100 μM at −20 °C until use.
DNA concentration was measured using a microvolume spectrometer (DS-11FX,
DeNovix, Wilmington, DE, USA). DNA sequences are provided in Table S1. Sorbitan monooleate (Span 80) and oleylamine
were purchased from Tokyo Chemical Industry (Tokyo, Japan) and Kanto
Chemical (Tokyo, Japan), respectively. Ultrapure water and Tris-HCl
(pH 8.0) were purchased from Thermo Fisher Scientific (Waltham, MA,
USA). NaCl powder, cholesterol, polyoxyethylene (10) octylphenyl ether
(Triton-X 100), sucrose, and liquid paraffin were purchased from FUJIFILM
Wako Pure Chemical (Osaka, Japan). Iodixanol (OptiPrep) was purchased
from Cosmo Bio (Tokyo, Japan). Silicone-coated glasses (30 mm by 40
mm with a thickness of 0.17 mm) were purchased from Matsunami Glass
(Osaka, Japan). 1,2-Dioleoyl-sn-glycero-3-phosphocholine
(DOPC) was purchased from NOF Corporation (Tokyo, Japan). 1,2-Dioleoyl-3-trimethylammonium-propane
(DOTAP) was purchased from Avanti Polar Lipids (Alabaster, AL, USA).
Exonuclease I and III were purchased from New England Biolabs (Ipswich,
MS, USA).

Sato Y, & Takinoue M. (2021). Capsule-like DNA Hydrogels with Patterns Formed by Lateral Phase Separation of DNA Nanostructures. JACS Au, 2(1), 159-168.

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Biotinylation of Serum Samples

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Biotinylation of serum was performed on ice or at 4 °C according to standard procedure. Briefly, serum samples were centrifuged at 16,000× g and the resulting cleared serum then diluted in PBS (D-PBS-Sterile w/o Mg, Ca, GE Healthcare Life Sciences, Marlborough, MA, USA) to 1:5 before the addition of biotin (EZ-LinkTM NHS-PEG4-Biotin, Thermo Fisher Scientific, Waltham, MA, USA) to a final concentration of 1.13 mM. The reaction was terminated after 2 h by adding Tris-HCl pH 8.0 (Thermo Fisher Scientific, Waltham, MA, USA) to a final concentration of 181 mM. For each biotinylation plate, three replicates of a reference serum sample (ERM-DA470k/IFCC, JRC, Geel, Belgium) were included as the process control. Biotinylated samples were pooled, aliquoted, and stored at −80 °C.

Verardo D., Liljedahl L., Richter C., Agnarsson B., Axelsson U., Prinz C.N., Höök F., Borrebaeck C.A, & Linke H. (2021). Fluorescence Signal Enhancement in Antibody Microarrays Using Lightguiding Nanowires. Nanomaterials, 11(1), 227.

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DNA Sample Preparation and Characterization

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Materials: DNAs were purchased from Eurofins Genomics (Tokyo, Japan). Non-modified and FAM-modified DNAs were of oligonucleotide purification cartridge grade, and Alexa 405-and Cy3modified DNAs were of high-performance liquid chromatography purification grade. DNA was stored in Ultrapure water (18 megaohms•cm in resistance) at a concentration of 100 µM at -20 °C until use. DNA concentration was measured using a microvolume spectrometer (DS-11FX, DeNovix, Wilmington, DE, USA). DNA sequences are provided in Table S1. Sorbitan monooleate (Span 80) and oleylamine were purchased from Tokyo Chemical Industry (Tokyo, Japan) and Kanto Chemical (Tokyo, Japan), respectively. Ultrapure water and Tris-HCl (pH 8.0) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). NaCl powder and liquid paraffin were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). Silicone-coated glasses (30 mm by 40 mm with a thickness of 0.17 mm) were purchased from Matsunami Glass (Osaka, Japan).

Sato Y., & Takinoue M. (2021). Pattern Regulation of DNA Hydrogels Formed by Lateral Phase Separation of DNA Nanostructures on Water-in-Oil Droplet Interfaces.

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Biomolecular Reagent Preparation Protocol

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MgCl2 (Mallinckrodt CHEMICALS, 6066-04), 100% Glycerol (Sigma-Aldrich, G5516), RNase/DNase-free UPW (Invitrogen, 10977-035), 1 M Tris–HCl pH 8.0 (Invitrogen, 15568–025), 1 M Tris–HCl pH 7.5 (Invitrogen, 15567-027), 0.5M EDTA (Invitrogen, AM9261), 1M DTT (Sigma-Aldrich, 43816), Tween-20 (Sigma-Aldrich, P1379), TBE ×10 (Fisher BioReagents, BP13334). Stock solutions of 4 M of NaCl (J.T. Baker, 0277), 3 M KCl (powder; MERCKGaA, 104936), 3 M LiCl (powder; J.T. Baker, 2370–01), 50mM ATP (Jena Bioscience, NU-1010) or 50 mM ATPgS (Jena Bioscience, NU-406) were prepared by dissolving the powders in RNase-free UPW or in UPW, treated with DEPC (Sigma-Aldrich, D5758) before use.

Danino Y.M., Molitor L., Rosenbaum-Cohen T., Kaiser S., Cohen Y., Porat Z., Marmor-Kollet H., Katina C., Savidor A., Rotkopf R., Ben-Isaac E., Golani O., Levin Y., Monchaud D., Hickson I.D, & Hornstein E. (2023). BLM helicase protein negatively regulates stress granule formation through unwinding RNA G-quadruplex structures. Nucleic Acids Research, 51(17), 9369-9384.

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Co-immunoprecipitation and GST Pulldown Assays

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293T cells were transfected with the indicated DNA plasmids using polyethylenimine transfection (Polysciences). Cells were collected after 48 h post-transfection, washed by PBS (Sigma) and resuspended in 1% NP40 lysis buffer containing 50 mM Tris-HCl, pH 8.0 (Invitrogen), 150 mM NaCl (Sigma) and 1% Nonidet P-40 (Sigma) supplemented with EDTA-free complete protease inhibitor cocktail (Roche). After sonication or three freeze/thaw cycles, WCL were centrifuged for 10 min at 16,000g. The supernatants, referred to as ‘WCEs’, were precleared with Sepharose beads (GE) rotating at 4 °C for 1 h and filtered through a 0.45 μm polyethersulfone filter (Thermo Fisher). For co-immunoprecipitation, WCEs were incubated with indicated antibodies at 4 °C for 3–12 h, followed by incubation with protein A/G agarose beads (Thermo Fisher) at 4 °C for 3 h. For GST pulldown, WCEs were incubated with glutathione-conjugated Sepharose beads (GE) at 4 °C for 1 h. The immobilized immunocomplexes or GST complexes containing beads were washed five times using 1% NP40 lysis buffer with various concentrations of NaCl (150–500 mM). Beads were eluted in 2× Laemmli dye (Sigma) by heating at 95 °C for 5 min and subjecting to immunoblotting.

Choi Y.J., Kim S., Choi Y., Nielsen T.B., Yan J., Lu A., Ruan J., Lee H.R., Wu H., Spellberg B, & Jung J.U. (2019). SERPINB1-mediated checkpoint of inflammatory caspase activation. Nature immunology, 20(3), 276-287.

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