Dithiothreitol (dtt)

Manufactured by New England Biolabs

Sourced in United States

DTT is a reducing agent commonly used in molecular biology applications. It helps maintain proteins in their reduced state by preventing oxidation. DTT is a small, water-soluble molecule that can be easily added to various biological buffers and solutions.

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Lab products found in correlation

Rotifree stripping buffer, by Carl Roth (2 mentions) Non fat dry milk, by Carl Roth (2 mentions) Phosphosafe lysis buffer, by Merck Group (2 mentions) Mini protean tgx any kd precast gels, by Bio-Rad (2 mentions) Amersham ecl prime detection reagent, by GE Healthcare (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Chemidoc xrs imaging system, by Bio-Rad (2 mentions) Dc protein assay, by Bio-Rad (2 mentions) Sds sample buffer, by New England Biolabs (2 mentions) Qiaquick gel extraction kit, by Qiagen (2 mentions) Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions) Cycloheximide (chx), by Merck Group (1 mentions) Mgcl2, by New England Biolabs (1 mentions) Maltoheptaose, by Merck Group (1 mentions) Peroxidase blocking reagent, by Agilent Technologies (1 mentions) Peptide n glycosidase f pngase f, by New England Biolabs (1 mentions) N n dimethylaniline, by Merck Group (1 mentions) Antigen retrieval buffer, by R&D Systems (1 mentions) Abc elite kit, by Vector Laboratories (1 mentions) 2 5 dihydroxybenzoic acid, by Merck Group (1 mentions)

21 protocols using dithiothreitol (dtt)

Western Blot Analysis of Phage and CHO Cells

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Phage was denatured via heating in SDS sample buffer containing 3× loading dye and DTT (NEB). Equivalent amounts of protein were resolved on 20% Tris glycine gels (Novex) and transferred onto poly(vinylidene difluoride) (PVDF) membranes (Millipore) for immunoblot analysis.
All CHO cell blots used cells grown to ~90% confluence in 6-well plates, treated with the indicated testing conditions, and lysed in 150 μL of lysis buffer (PBS plus 1% Triton X-100, 1× protease/phosphatase inhibitor (CST)). Loading dye (3×) plus DTT (NEB) was added before heat denaturation, and samples were run on 4–15% TGX gels (Bio-Rad). Proteins were transferred to PVDF membranes and incubated with the appropriate antibodies. Quantitative western blots were performed using fluorescent secondary antibodies (800 and 680 nm, Li-Cor) and a Li-Cor Odyssey imager. Quantification was performed using the manufacturer’s software.

Beech J., Saleh L., Frentzel J., Figler H., Corrêa IR J.r., Baker B., Ramspacher C., Marshall M., Dasa S., Linden J., Noren C.J, & Kelly K.A. (2015). Multivalent Site-Specific Phage Modification Enhances the Binding Affinity of Receptor Ligands. Bioconjugate chemistry, 26(3), 529-536.

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Detergent-Based Protein Fractionation Assay

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For the solubility assays, cell lysis and protein fractionation based on detergent solubility were performed as previously described (Figure 2A; Guo and Lee, 2011 (link); Kfoury et al., 2012 (link); Silva et al., 2016 (link)). Briefly, higher solubility proteins (S fractions) were purified in 1% Triton buffer [1% Triton X-100 (Thermo Fisher Scientific), 1% Halt Protease/Phosphatase inhibitors (Thermo Fisher Scientific), 1:5,000 Benzonase (Sigma) and 10 mM DTT (New England BioLabs) in DPBS], whereas lower solubility pelleted proteins (P fractions) were resuspended in 5% SDS buffer [5% SDS (Sigma), 1% Halt Protease/Phosphatase inhibitors (Thermo Fisher Scientific), 1:5,000 Benzonase (Sigma) and 10 mM DTT (New England BioLabs) in RIPA buffer]. SDS-PAGE western blot was performed by loading 20 μg of each S-fraction and equal volume of the P-fraction onto pre-cast Tris-Acetate SDS-PAGE (Novex, Invitrogen). Western blot was performed as before. Densitometry values (pixel mean intensity in arbitrary units, a.u.) were measured with the Histogram function of Adobe Photoshop 2021 (v.22.4.3) and normalized to the respective GAPDH intensity in the S-fraction. Calculations were done in Microsoft Excel (v.16.52) and graphs were plotted in GraphPad Prism 9 (v.9.2.0).

Silva M.C., Nandi G., Donovan K.A., Cai Q., Berry B.C., Nowak R.P., Fischer E.S., Gray N.S., Ferguson F.M, & Haggarty S.J. (2022). Discovery and Optimization of Tau Targeted Protein Degraders Enabled by Patient Induced Pluripotent Stem Cells-Derived Neuronal Models of Tauopathy. Frontiers in Cellular Neuroscience, 16, 801179.

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Yeast Cell Lysis for Translational Profiling

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Yeast cells were collected on 0.45 μm pore size membrane filters (Millipore, Cat. # HAWP04700) via filtration, immediately removed with a cell scraper (TPP, Cat. # 99002, TPP Techno Plastic Products AG, Trasadingen, Schaffhausen, Switzerland), then placed into Nunc 50 mL conical sterile polypropylene centrifuge tubes (Thermo Fisher Scientific, cat. No. 339652) containing liquid nitrogen. Then, 0.6 mL lysis buffer (20 mM Tris-HCl pH 7.5 (Wako Pure Chemical Industries, Ltd., Cat. # 318-90225, Wako Pure Chemical Industries, Ltd., Osaka, Japan), 150 mM NaCl (Nacalai Tesque, Cat. # 06900-14, NACALAI TESQUE, Inc., Kyoto, Japan), 5 mM MgCl2 (Nacalai Tesque, Cat. # 20942-34), 1 mM dithiothreitol (New England Biolabs, Cat. # M0368L, New England Biolabs Inc., Ipswich, MA, USA), 100 µg/mL cycloheximide (Sigma-Aldrich, Cat. # C4859-1ML), 100 µg/mL chloramphenicol (Wako Pure Chemical Industries, Cat. # 030-19452), and 1% Triton X-100 (Nacalai Tesque, Cat. # 12967-32)) was added dropwise into the tubes to obtain flash-frozen droplets. Finally, the tubes were stored at −80 °C until the liquid nitrogen evaporated.

Zhao T., Chida A., Shichino Y., Choi D., Mizunuma M., Iwasaki S, & Ohya Y. (2022). Multifarious Translational Regulation during Replicative Aging in Yeast. Journal of Fungi, 8(9), 938.

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Radiation Effects on BamHI Endonuclease Activity

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Pre- and postirradiation activity of BamHI was determined as described previously (15 (link), 20 (link)) with some modifications. BamHI (5,000 U/μL) (without bovine serum albumin [BSA]) (New England Biolabs, Ipswich, MA, USA) was diluted in D. radiodurans or in C. elegans ultrafiltrate to 0.54 U/μL. Then, 200 μL of the BamHI mixture was gamma-irradiated aerobically on ice. Following irradiation, 20 μL of each radiation-treated BamHI sample was assayed for residual endonuclease activity in separate reaction mixtures (final volume, 30 μL) containing 125 ng lambda phage DNA, 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl₂, and 1 mM dithiothreitol (New England Biolabs; buffer 3). BamHI/lambda DNA mixtures were incubated for 1.25 h at 37°C, followed by agarose (0.8%) gel electrophoresis.

Gaidamakova E.K., Sharma A., Matrosova V.Y., Grichenko O., Volpe R.P., Tkavc R., Conze I.H., Klimenkova P., Balygina I., Horne W.H., Gostinčar C., Chen X., Makarova K.S., Shuryak I., Srinivasan C., Jackson-Thompson B., Hoffman B.M, & Daly M.J. (2022). Small-Molecule Mn Antioxidants in Caenorhabditis elegans and Deinococcus radiodurans Supplant MnSOD Enzymes during Aging and Irradiation. mBio, 13(1), e03394-21.

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Glycosylation Analysis of Proteins

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Antigen retrieval buffer was
purchased from R&D Systems. Peptide-N-Glycosidase F (PNGase F)
was from New England Biolabs, dithiothreitol (DTT), maltoheptaose
(DP7), 2,5-dihydroxybenzoic acid (DHB), and N,N-Dimethylaniline
(DMA) were purchased from Sigma-Aldrich. Biotinylated AAL and ConA
lectins and the ABC-Elite kit were from Vector Laboratories. Peroxidase
blocking reagent was from Dako.

Toghi Eshghi S., Yang S., Wang X., Shah P., Li X, & Zhang H. (2014). Imaging of N-Linked Glycans from Formalin-Fixed Paraffin-Embedded Tissue Sections Using MALDI Mass Spectrometry. ACS Chemical Biology, 9(9), 2149-2156.

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Protein Expression and Western Blot Analysis

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Confluent cells were washed once with ice-cold PBS and lysed with 1 × PhosphoSafe lysis buffer (Merck Millipore). Cell lysates were sonicated and centrifuged for 10 min at 14,000× g and 4 °C following determination of total protein concentration in supernatants using BioRad DC protein assay. Samples containing equal amounts of protein were prepared using 3× SDS sample buffer and 125 mM dithiothreitol (both from New England Biolabs, Ipswich, MA, USA), subjected to gel electrophoresis using BioRad mini-PROTEAN TGX any-kD precast gels and blotted to 0.45 μm PVDF membranes. Membranes were blocked with nonfat dry milk (Carl Roth GmbH, Karlsruhe, Germany) or BSA (Sigma-Aldrich) and incubated with primary antibodies at 4 °C overnight. HRP-linked secondary antibodies and Amersham ECL Prime Detection Reagent (GE Healthcare Dharmacon) were used for detection of proteins on a BioRad ChemiDoc XRS imaging system. RotiFree stripping buffer (Carl Roth GmbH) was used for membrane stripping.

Melzer C., von der Ohe J., Hass R, & Ungefroren H. (2017). TGF-β-Dependent Growth Arrest and Cell Migration in Benign and Malignant Breast Epithelial Cells Are Antagonistically Controlled by Rac1 and Rac1b. International Journal of Molecular Sciences, 18(7), 1574.

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Western Blot Protein Analysis Protocol

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Confluent cells were washed once with ice-cold PBS and lysed with 1x PhosphoSafe lysis buffer (Merck Millipore). Cell lysates were sonicated and centrifuged for 10 min at 14.000 × g and 4 °C following determination of total protein concentration in supernatants using BioRad DC Protein Assay. Samples containing equal amounts of protein were prepared using 3x SDS Sample Buffer and 125 mM Dithiothreitol (both from New England Biolabs), subjected to gel electrophoresis using BioRad mini-PROTEAN TGX any-kD precast gels and blotted to 0.45 μm PVDF membranes. Membranes were blocked with nonfat dry milk (Carl Roth GmbH, Karlsruhe, Germany) or BSA (Sigma-Aldrich) and incubated with primary antibodies at 4 °C overnight. HRP-linked secondary antibodies and Amersham ECL Prime Detection Reagent (GE Healthcare) were used for detection of proteins on a BioRad ChemiDoc XRS imaging system. RotiFree stripping buffer (Carl Roth GmbH) was used for membrane stripping. Signal intensities were quantified by densitometry and computed with either NIH image J or Image Lab (version 5.2.1, BioRad).

Witte D., Otterbein H., Förster M., Giehl K., Zeiser R., Lehnert H, & Ungefroren H. (2017). Negative regulation of TGF-β1-induced MKK6-p38 and MEK-ERK signalling and epithelial-mesenchymal transition by Rac1b. Scientific Reports, 7, 17313.

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Quantitative RT-PCR Protocols for Plant RNAs

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RT-PCRs were performed in 2 steps. For the first cDNA synthesis, 100 ng total RNA, extracted from the latex of fruits or from plant tissues, was mixed with either 1.5 μM specific primers or an oligo-dT/random hexamer mix adjusted to 10 μL with ddH 2 O and incubated at 70°C for 5 min. Next, the RNA/primer mix was used in 20-μL reactions containing 10 U M-MuLV reverse transcriptase, 1X M-MuLV RT buffer, 0.25 mM dNTPs, and 0.25 mM dithiothreitol (all reagents from New England Biolabs). The reactions were incubated for 1 h at 42°C, 15 min at 70°C, and adjusted to a final volume of 50 μL. For second-strand synthesis, 50 μL PCRs contained 0.2 μM specific primers (Table 1), 0.2 mM dNTPs, 1.25 U Taq polymerase, 1X Thermo Pol buffer, and 2 μL first-strand cDNA. Reactions were performed using the following program: 94°C for 4 min, 30 cycles of 30 s for 94°C, 52°-60°C for 30 s, 72°C for 1 min, and a 10-min final extension at 72°C. Next, 5 μL was loaded onto a 0.8% agarose gel containing ethidium bromide. To assess the limit of detection (LOD), serial 10-fold dilutions of total RNA were used as a template in 25-μL 1-step RT-PCRs. The PCR products were purified using the QIAquick PCR purification kit (Qiagen) and ligated into the pGEMT-easy vector. Plasmid DNA from 3 positive clones was sequenced as described above.

Zamudio-Moreno E., Ramirez-Prado J.H., Moreno-Valenzuela O.A, & Lopez-Ochoa L.A. (2015). Early diagnosis of a Mexican variant of Papaya meleira virus (PMeV-Mx) by RT-PCR. Genetics and molecular research : GMR, 14(1).

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Reverse Transcription Protocol for cDNA Synthesis

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A total of 0.7 μl RT Primer (from a 100 μM stock) was added to the purified sample and incubated at 75°C for 3 min, 37°C for 10 min and 25°C for 10 min. A total of 4 μl of ProtoScript II Buffer, 1 μl dNTP mix (NEB), 0.2 μl 1 M MgCl₂, 2 μl DTT (NEB) and 2 μl of ProtoScript II were added and the mixture incubated at 42°C for 12 h with a 105°C heated lid. (Alternatively, for standard conditions [see section on 2′-5′ linkages], no MgCl₂ was added and the incubation was at 50°C for 1 h.) Ten microliter of water was added and the mixture was run through an Oligo Clean & Concentrator spin column, including the RNA degradation step as per the manufacturer's instructions. The cDNA was eluted in 30 μl of TE, pH 7 and stored at 4°C. The cDNA stock concentration was measured with a NanoDrop 2000c spectrophotometer (ThermoFisher Scientific), and typically found to be in the fraction of a μg/μl range.

Duzdevich D., Carr C.E, & Szostak J.W. (2020). Deep sequencing of non-enzymatic RNA primer extension. Nucleic Acids Research, 48(12), e70.

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SARS-CoV-2 Nucleocapsid Protein ELISA

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The native recombinant nucleocapsid proteins (NPs) of SARS-CoV, SARS-CoV-2 or variants, MERS-CoV, HKU1, OC43, NL63, and 229E (Sino Biological) (2 μg/mL) or denatured by treatment with the denaturing buffer containing 0.5% SDS and 40 mM DTT [18 (link)] (New England Biolabs) at 95 °C for 10 min were coated into 96-well half-area plates overnight at 4 °C. The plates were then blocked with blocking buffer (PBS containing 4% skim milk) at 37 °C for 1 h. Five-fold or three-fold serial-diluted NP-specific mAbs or Rabbit pAb against NP of SARS-CoV-2 (Sino Biological) were added to the plates in duplicate and incubated for 1 h at 37 °C. And then HRP-conjugated Goat anti-Human IgG (ZSGB-BIO) or Goat anti-Rabbit IgG (TransGen Biotech) secondary antibody was added to the plates and incubated at 37 °C for 1 h. For competitive ELISA, after blocking, serially diluted P301-F7, P301-H5, and human IgG1 were mixed with HRP-conjugated P301-F7, and then added to the plates and incubated at 37 °C for 1 h. The enzymatic reaction was developed with TMB substrate (BD Biosciences) and stopped by 2 M H₂SO₄. The optical density was measured at 450 nm (OD. 450 nm) with a Varioskan™ LUX Multimode Microplate Reader (Thermo Scientific).

Zhou B., Cheng L., Song S., Guo H., Shen S., Wang H., Ge X., Liu L., Ju B, & Zhang Z. (2022). Identification and application of a pair of noncompeting monoclonal antibodies broadly binding to the nucleocapsid proteins of SARS-CoV-2 variants including Omicron. Virology Journal, 19, 96.

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