Dithiothreitol (dtt)

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DTT (Dithiothreitol) is a reducing agent commonly used in biochemistry and molecular biology applications. It is a small, water-soluble molecule that helps maintain the reduced state of cysteine residues in proteins. DTT is often used in protein purification, gel electrophoresis, and other procedures where the preservation of protein structure and function is essential.

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766 protocols using dithiothreitol (dtt)

Tau Aggregation Inhibition Assay

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Recombinant human P301L tau was dialyzed into 100 mM sodium acetate buffer pH 7.4 overnight. Samples consisting of 20 μM P301L tau and 2x the indicated concentration of compound were incubated at room temperature for 1 hour prior to starting the assay. Using a master mix of 2x concentrated heparin (MP Biomedicals, Santa Anna, CA), Thioflavin T (Sigma-Aldrich, St. Louis, MO), and dithiothreitol (ThermoFisher Scientific, Waltham, MA), samples were diluted to a final concentration of 10 μM P301L tau, 2.5 μM heparin, 10 μM Thioflavin T, the compounds at indicated concentrations or DMSO, and 2 mM dithiothreitol in 100 mM sodium acetate at pH 7.4. A total of 200 μL was added to each well of a low binding 96-well black sided clear bottom costar plate (Corning, Corning, NY). Each plate was incubated at 37 °C, and fluorescence intensity (440 excitation, 482 emission) was measured over a 5-day period using a BioTek (Winooski, VT) Synergy H1 microplate reader.

Hill S.E., Beaulieu-Abdelahad D., Lemus A., Webster J.M., Ospina S.R., Darling A.L., Martin M.D., Patel S., Bridenstine L., Swonger R., Paul S., Blackburn R., Calcul L., Dickey C.A., Leahy J.W, & Blair L.J. (2023). Benzothiazole Substitution Analogs of Rhodacyanine Hsp70 Inhibitors Modulate Tau Accumulation. ACS chemical biology, 18(5).

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Tau Aggregation Inhibition Assay

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Stadler J., Trockfeld J., Schmalix W.A., Brill T., Siewert J.R., Greim H, & Doehmer J. (1994). Inhibition of cytochromes P4501A by nitric oxide.. Proceedings of the National Academy of Sciences of the United States of America, 91(9), 3559-3563.

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SDS-PAGE Protein Analysis Protocol

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For SDS-PAGE analysis, precast 4–20% Bis-Tris gradient gels (Genscript, Picastaway, USA) in a Tris-MOPS running buffer was used. The molecular weight marker was Pierce™ Unstained MW marker (Thermo Scientific). Maximum volume of 20 µL was loaded in the wells on the SDS-PAGE gel. Solubilized extracts were mixed 14:5:1 with a 4xSDS sample buffer (50 mM Tris (VWR, Leuven, Belgium) pH 6.8, 2% SDS, 10% glycerol (VWR), 0.02% bromophenol blue (Sigma-Aldrich), 12.4 mM ethylenediaminetetraAcetic acid (EDTA, Carl Roth, Karlsruhe, Germany)) and 1 M dithiothreitol (DTT, Thermo Scientific), corresponding to a final DTT concentration of 50 mM. The samples incubated at 95 °C for 5 min to denature proteins. Running time for the gel was 45 min at 140 V. To visualize proteins, the gel was stained with Coomassie Brilliant Blue G250 (29 mM Coomassie Brilliant Blue G-250 (Sigma-Aldrich), 45% Ethanol (VWR), 10% Acetic acid (Fisher Scientific, Loughborough, UK)). Destaining of the gel was performed overnight with destain solution (8% Ethanol, 5% Acetic acid). Imaging of the gel was done using a ChemDoc MP Imaging System (Bio-Rad, Hercules, USA).

Gregersen S., Kongsted A.S., Nielsen R.B., Hansen S.S., Lau F.A., Rasmussen J.B., Holdt S.L, & Jacobsen C. (2021). Enzymatic extraction improves intracellular protein recovery from the industrial carrageenan seaweed Eucheuma denticulatum revealed by quantitative, subcellular protein profiling: A high potential source of functional food ingredients. Food Chemistry: X, 12, 100137.

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Infection Assay for Airway Epithelial Cells

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Primary human nasal epithelial cells (PromoCell) were pretreated with compounds for 3 hours, infected with virus at MOI=3 and incubated with medium containing 2 μM inhibitors for 48 hours, and fixed with 4% paraformaldehyde in PBS.
Primary human bronchial epithelial cells were cultured on 6.5-mm transwell inserts (Corning, Corning, NY, USA) for 4 weeks at air-liquid interface to obtain a well-differentiated epithelium. Mucus was removed immediately prior to viral infection by washing the apical surface with a prewarmed solution of 10 mM dithiothreitol (DTT; Thermo) in PBS for 10 minutes and then washed twice with PBS without DTT. Cells were inoculated by adding virus to the apical surface at MOI=1. After 1 h, the apical surface was washed twice with PBS and cells were returned to the incubator. Cells were fixed at 72 h post-infection in 4% paraformaldehyde in PBS.

Yang J., Xiao Y., Lidsky P.V., Wu C.T., Bonser L.R., Peng S., Garcia-Knight M.A., Tassetto M., Chung C.I., Li X., Nakayama T., Lee I.T., Nayak J.V., Ghias K., Hargett K.L., Shoichet B.K., Erle D.J., Jackson P.K., Andino R, & Shu X. (2023). Fluorogenic reporter enables identification of compounds that inhibit SARS-CoV-2. Nature microbiology, 8(1), 121-134.

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Oligonucleotide Reduction and Purification

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Oligonucleotide stock solution was added to dithiothreitol (DTT) (Acros Organics, Thermo Fisher Scientific, NJ, USA) in 20 mM sodium phosphate buffer at pH 7.4, bringing [DTT]_final to 100 mM, and [oligo]_final to 10 μM. The reduction reaction was allowed to proceed at room temperature protected from light for 60 minutes. DTT was removed by buffer exchange using a 15 mL, 3 kD molecular weight cutoff spin concentrator according to manufacturer’s instructions (EMD Millipore, Billerica, MA). In brief, the reaction solution was added to the reservoir of the spin concentrator and brought to 15 mL with 20 mM pH 7.4 sodium phosphate buffer. The concentrator was spun at 4000 × g for 30 minutes until approximately 500 μL was concentrated in the reservoir. The flow-through was then discarded, and the reservoir volume was brought back up to 15 mL with fresh 20 mM sodium phosphate buffer. The concentrator was spun for a total of four buffer exchange cycles. The resulting 500 μL concentrate was collected for analysis by mass spectrometry before proceeding with the next step of the synthesis.

Dempsey M.E., Marble H.D., Shen T.L., Fawzi N.L, & Darling E.M. (2018). Synthesis and characterization of a magnetically active 19F molecular beacon. Bioconjugate chemistry, 29(2), 335-342.

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Purification of p300-CH1-GST Fusion Protein

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Example 3

The pGEX-4T-2-p300 fusion vector was transformed into BL21(DE3) competent E.coli (Novagen) in M9 minimal media with ¹⁵NH₄Cl as the primary nitrogen source. Protein production was induced with 1 mM IPTG at OD₆₀₀of 1 for 16 hours at 15° C. Production of the desired p300-CH1-GST fusion product was verified by SDS-PAGE. Bacteria were harvested and resuspended in the lysis buffer with 20 mM Phosphate buffer (Research Products International, Corp.), 100 μM DTT (Fisher), 100 μM ZnSO₄(Sigma), 0.5% TritonX 100 (Sigma), 1 mg/mL Pepstatin A (Research Products International, Corp.), 10 mg/mL Leupeptin A (Research Products International, Corp.), 500 μM PMSF (Sigma), and 0.5% glycerol at pH 8.0. Pellets were lysed by sonication and centrifuged at 4° C. and 20,000 rpm for 20 minutes. Fusion protein was collected from the bacterial supernatant and purified by affinity chromatography using glutathione Sepharose 4B beads (Amersham) prepared according to the manufacturer's directions. GST-tag was cleaved by thrombin and protein was eluted from resin. Collected fractions were assayed by SDS-PAGE gel; pooled fractions were treated with protease inhibitor cocktail (Sigma) and against a buffer containing 10 mM Tris, 50 mM NaCl, 2 mM DTT (Fisher), and 3 equivalents ZnSO₄at pH 8.0 to ensure proper folding (vide supra).

US10787424B2. Oxopiperazine helix mimetics for control of Hypoxia-Inducible gene expression (2020-09-29). NEW YORK UNIVERSITY [US]. Inventors: Paramjit S. Arora [US], Brooke Bullock Lao [US], Richard Bonneau [US], Kevin Drew [US].

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Purification of p300-CH1-GST Fusion Protein

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Example 3

The pGEX-4T-2-p300 fusion vector was transformed into BL21(DE3) competent E. coli (Novagen) in M9 minimal media with ¹⁵NH₄Cl as the primary nitrogen source. Protein production was induced with 1 mM IPTG at OD₆₀₀of 1 for 16 hours at 15° C. Production of the desired p300-CH1-GST fusion product was verified by SDS-PAGE. Bacteria were harvested and resuspended in the lysis buffer with 20 mM Phosphate buffer (Research Products International, Corp.), 100 μM DTT (Fisher), 100 μM ZnSO₄(Sigma), 0.5% TritonX 100 (Sigma), 1 mg/mL Pepstatin A (Research Products International, Corp.), 10 mg/mL Leupeptin A (Research Products International, Corp.), 500 μM PMSF (Sigma), and 0.5% glycerol at pH 8.0. Pellets were lysed by sonication and centrifuged at 4° C. and 20,000 rpm for 20 minutes. Fusion protein was collected from the bacterial supernatant and purified by affinity chromatography using glutathione Sepharose 4B beads (Amersham) prepared according to the manufacturer's directions. GST-tag was cleaved by thrombin and protein was eluted from resin. Collected fractions were assayed by SDS-PAGE gel; pooled fractions were treated with protease inhibitor cocktail (Sigma) and against a buffer containing 10 mM Tris, 50 mM NaCl, 2 mM DTT (Fisher), and 3 equivalents ZnSO₄at pH 8.0 to ensure proper folding (vide supra).

US11560359B2. Oxopiperazine helix mimetics for control of hypoxia-inducible gene expression (2023-01-24). NEW YORK UNIVERSITY [US]. Inventors: Paramjit S. Arora [US], Brooke Bullock Lao [US], Richard Bonneau [US], Kevin Drew [US].

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SARS-CoV-2 Infection of Human Airway Epithelia

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SARS-CoV-2 virus (USA-WA1/2020 strain) was provided by Dr. Melanie Ott and propagated in Vero E6 cells. HBECs were cultured in the absence or presence of IL-13 (10 ng/ml). Mucus was allowed to accumulate for 3 days or was removed immediately before viral infection by washing the apical surface with a prewarmed solution of 10 mM DTT (Thermo) in PBS for 10 minutes (26 (link)) and then washing twice with PBS without DTT. Cells were inoculated by adding virus to the apical surface. After 2 hours, the apical surface was washed twice with PBS, and cells were returned to the incubator. Cells were harvested 48 hours after infection for analysis of viral RNA (by qRT-PCR) and double-stranded RNA (dsRNA) staining (by immunofluorescence).

Bonser L.R., Eckalbar W.L., Rodriguez L., Shen J., Koh K.D., Ghias K., Zlock L.T., Christenson S., Woodruff P.G., Finkbeiner W.E, & Erle D.J. (2022). The Type 2 Asthma Mediator IL-13 Inhibits Severe Acute Respiratory Syndrome Coronavirus 2 Infection of Bronchial Epithelium. American Journal of Respiratory Cell and Molecular Biology, 66(4), 391-401.

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Protein Expression Analysis of Explanted mSVF

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Explanted mSVF was homogenized by sonication and lysates were acquired by lithium dodecyl sulfate (LDS)/ dithiothreitol (DTT) buffer LDS/DTT buffer (25% (v/v) NuPAGE lithium LDS sample buffer (Invitrogen, Karlsruhe, Germany), 50 mM DTT (Sigma-Aldrich, Munich, Germany), bidistilled water (ddH₂O)). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for Akt (55 kDa), pAkt (56–60 kDa), ERK/pERK-1/2 (42–44 kDa), actin (43 kDa) and α-tubulin were performed on 10% polyacrylamide gels. For Western blotting, the primary antibodies Akt (Abcam, Cambridge, UK), pAkt (Abcam, Cambridge, UK), ERK1/2 (Cell Signaling Technology, Danvers, MA, USA), pERK1/2 (Cell Signaling Technology, Danvers, MA, USA), actin (Merck& Co., Kenilworth, NJ, USA), α tubulin (Abcam, Cambridge, UK) and the secondary antibodies HRP-goat anti-mouse IgG, HRP-goat anti-rabbit IgG and HRP-rabbit anti-rat IgG from Abcam, Cambridge, UK were used. HRP on the blots was detected by ECL substrate (Bio-Rad Laboratories, Hercules, CA, USA).

Kim B.S., Chen S.H., Vasella M., Guidi M., Gousopoulos E., Lindenblatt N, & Kao H.K. (2022). In Vivo Evaluation of Mechanically Processed Stromal Vascular Fraction in a Chamber Vascularized by an Arteriovenous Shunt. Pharmaceutics, 14(2), 417.

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Isolation of Intestinal Epithelial and Lamina Propria Lymphocytes

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Steady-state intestinal epithelial and lamina propria lymphocytes were harvested as previously described^{49 (link)}. To isolate IECs, intestinal tissues were first incubated in 5 mM EDTA (Invitrogen) in HBSS (Gibco) containing 1 mM DTT (Invitrogen) for 20 minutes at 37°C with 200rpm agitation, and then incubated in a second wash of 5 mM EDTA in HBSS without DTT for another 20 minutes at 37°C with agitation. Single-cell suspensions released into the EDTA solutions were pooled and confirmed to contain over 85% EpCAM⁺ IECs as reported previously^{39 (link)}. For the lamina propria, IEC-depleted tissues from post-EDTA washes were digested in a HBSS solution containing 10% Fetal Bovine Serum (Peak Serum), 1.0 mg/ml Collagenase D (Roche), 100 μg/ml DNase I (Sigma-Aldrich), and 50 U/ml dispase (Worthington Biochemical Corporation) at 37°C for 30–45 minutes. Lamina propria mononuclear lymphocytes were purified from the interphase of a 40:80% Percoll^™ (Cytiva; formerly GE Healthcare Life Sciences) gradient.

Long T., Abbasi N., Hernandez J.E., Li Y., Sayed I.M., Ma S., Iemolo A., Yee B.A., Yeo G.W., Telese F., Ghosh P., Das S, & Huang W.J. (2021). RNA binding protein DDX5 directs tuft cell specification and function to regulate microbial repertoire and disease susceptibility in the intestine. Gut, 71(9), 1790-1802.

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