Dithiothreitol (dtt)

Manufactured by Cytiva

Sourced in United States

DTT, or Dithiothreitol, is a reducing agent commonly used in biochemical applications. It functions by breaking down disulfide bonds in proteins, which can be useful for maintaining protein structure and activity during various experimental procedures.

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4 protocols using dithiothreitol (dtt)

Comprehensive Immunoblotting Profiling of Cellular Signaling

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Five-7 x 10⁶ cells were prepared by lysis in a buffer made up of 20 mM Tris, 150 mM NaCl, 2 mM EDTA (ethylenediaminetetraacetic acid), 2 mM EGTA (ethylene glycol-tetra-acetic acid), 0.5% v/v Triton X-100 supplemented with protease inhibitor cocktail (Sigma), 1 mM DTT (dithiothreitol; Amersham Biosciences), 1 mM PMSF (phenyl-methyl-sulfonyl fluoride; Sigma), 1 mM okadaic acid (Sigma) and phosphatase inhibitor cocktail (Thermo Scientific). Proteins were subjected to SDS-PAGE, transferred to PVDF membranes and immunoblotted with the following primary Abs: anti-CK2α (provided by Dr. M. Ruzzene, University of Padova, Italy), anti-CK2β, anti-RELA, anti-FOXO1 (Abcam), anti-pRELA S529 (recognizes S527 in mouse), anti-IRF4, anti-BLIMP-1, anti-BCL6 (Santa Cruz), anti-GAPDH (Ambion), anti-pAKT S129 (provided by Dr. M. Ruzzene, University of Padova, Italy), anti-AID (Invitrogen), anti-NOTCH2 (D76A6), anti-pAKT S473, anti-AKT, anti-pERK1/2 T202/Y204, anti-pBTK Y223, anti-ERK1/2, anti-pPTEN S380/T382/T383, PTEN, anti-pFOXO1 S256, anti-pSTAT6 Y705, anti-STAT6 (Cell Signaling). Secondary Abs: anti-rabbit IgG HRP-linked Ab (Cell Signaling), HRP labeled goat anti-mouse IgG (KPL), goat anti-rat IgG HRP-conjugated (Calbiochem), donkey anti-goat IgG HRP-conjugated (Santa Cruz).

Quotti Tubi L., Mandato E., Canovas Nunes S., Arjomand A., Zaffino F., Manni S., Casellato A., Macaccaro P., Vitulo N., Zumerle S., Filhol O., Boldyreff B., Siebel C.W., Viola A., Valle G., Mainoldi F., Casola S., Cancila V., Gulino A., Tripodo C., Pizzi M., Dei Tos A.P., Trentin L., Semenzato G, & Piazza F. (2023). CK2β-regulated signaling controls B cell differentiation and function. Frontiers in Immunology, 13, 959138.

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Affinity Purification of GST-Tagged Proteins

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Epoxy activated sepharose 6B and glutathione (GSH) were purchased from sigma chemical co. (St. louis, MO, USA). IPG strips, IPG buffer, DTT, CHAPS and urea were obtained from Amersham biosciences, NJ, USA. Rabbit polyclonal anti-GST antibody, Goat anti- rabbit IgG ALP conjugate secondary antibody, BCIP-NBT substrate for alkaline phosphatase and protein molecular weight marker were obtained from Bangalore Genei, India. All other chemicals were of analytical grade and were obtained from local firms.

Gopi S., Vadde R, & Setty O.H. (2017). Protective effect of aqueous extract of Phyllanthus fraternus against bromobenzene induced changes on cytosolic glutathione S-transferase isozymes in rat liver. Biochemistry and Biophysics Reports, 10, 208-214.

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Purification and characterization of protease inhibitors

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R. sublobata (Schumach.) Meikle (ICP 15868) seeds were obtained from International Crop Research Institute for Semi-Arid Tropics (ICRISAT), Hyderabad, India. N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA), N-glutaryl-L-phenylalanine-p-nitroanilide (GLUPHEPA) and soybean BBI were supplied by Sigma-Aldrich, USA. CNBr activated Sepharose, Sephadex G-50 fine and DTT were procured from GE Healthcare Biosciences Corp., USA. 3X crystals of bovine pancreatic αtrypsin and α-chymotrypsin, and all other chemicals were procured from Sisco Research Laboratories, Mumbai, India. H. armigera eggs were procured from National Bureau of Agricultural Insect Resources (NBAIR), Bengaluru, India. A. janata larvae were collected from the surrounding fields of the University of Hyderabad. The insects were reared in insect culture room maintained at temperature of 26 ± 1 °C, RH of 65 ± 5% and 14:10 h light-dark photoperiod.

Mohanraj S.S., Gujjarlapudi M., Lokya V., Mallikarjuna N., Dutta-Gupta A, & Padmasree K. (2019). Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea. Phytochemistry, 159.

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Purification and Characterization of Proteases

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Bovine serum albumin, bovine α-trypsin, α-chymotrypsin, casein and Polyvinylpyrrolidone (PVP) were procured from Sisco Research Laboratory, Mumbai, India. CNBr activated Sepharose 4B, Sephadex G-50 fine, BAPNA, GLUPHEPA, soybean trypsin-CI (soybean BBI), tricine, gelatin, phenylmethylsulfonyl fluoride (PMSF), Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), Np-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), Trizol reagent and Coomassie brilliant blue R-250 were purchased from Sigma Aldrich, United States. Immobiline pH gradient dry strips (IPG strips), IPG buffer, DTT, IDA, urea, thiourea, 3-[(3-Cholamidopropyl) dimethylammonio]-1-Propanesulfonate hydrate (CHAPS), CM5 sensor chips, amine coupling kit and HBS EP + 10X buffer were procured from GE Healthcare Biosciences Corp., United States. Verso cDNA synthesis kit, 50 bp DNA ladder, bicinchoninic acid (BCA) protein estimation kit, protein molecular mass standard, and 3 kDa cut-off SnakeSkin dialysis membrane were purchased from Thermo Fisher Scientific, United States. SYBR Green PCR Master Mix purchased from Takara Bio, Shiga, Japan and all other PCR components from New England Biolabs. Amicon ultra centrifugal filter units were purchased from Millipore Corporation, United States and all other chemicals and reagents used were of analytical grade.

Lokya V., Swathi M., Mallikarjuna N, & Padmasree K. (2020). Response of Midgut Trypsin- and Chymotrypsin-Like Proteases of Helicoverpa armigera Larvae Upon Feeding With Peanut BBI: Biochemical and Biophysical Characterization of PnBBI. Frontiers in Plant Science, 11, 266.

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