Dithiothreitol, DTT

The carbamate, prepared from the 4-nitrophenyl carbonate, is cleaved by reduction with dithiothreitol (DTT) and TEA to give the aniline, which triggers fragmentation, releasing the amine. ... 

From Sigma 3-aminoethylcarbazole (AEC) acrylamide/bis-acrylamide (30%) 37.5 1 amino acids alumina bentonite benzamidine bovine fiver tRNA bovine serum albumin (BSA) creatine phosphate (CP) diethyl pyrocarbonate (DEPC) dithiothreitol (DTT) Escherichia coli MRE600 tRNA pyrophosphatase (Ppase) Ca++ salt of folinic acid, (5-formyl THF) IIHPHS K salt of phospho-enol pyruvic acid, (PEP) creatine phospho kinase (CPK) protease inhibitor cocktail for fungal and yeast extracts phenylmethylsulfonyl fluoride (PMSF) spermidine trihydrochloride Tween 20. 

A study of by Palmer-Toy et al.,12 summarized in Table 19.1, provides further empirical evidence of the utility of techniques coupling heating with efficient protein extraction for the proteomic analysis of FFPE tissue. A specimen from a patient with chronic stenosing external otitis was divided in half and preserved as fresh-frozen tissue or FFPE. Ten micromolar sections of the FFPE tissue were vortexed in heptane to deparaffinize the tissue and were then co-extracted with methanol. The methanol layer was evaporated, and the protein residue was resuspended in 2% SDS/lOOmM ammonium bicarbon-ate/20mM dithiothreitol (DTT), pH 8.5 and heated at 70°C for lh. After tryptic digestion, 123 total confident proteins were identified in the FFPE tissue, compared to 94 proteins identified from the fresh-frozen tissue. Hwang et al. also reported up to a fivefold increase in protein extraction efficiency for samples extracted in a Tris-HCl/2% SDS/1% Triton X-100/1% deoxycholate solution at 94°C for 30 min versus samples extracted in 100 mM ammonium bicarbonate/30% acetonitrile at the same temperature.14... 

Dithiothreitol (DTT) and dithioerythritol (DTE) are the trans and cis isomers of the compound 2,3-dihydroxy-1,4-dithiolbutane. The reducing potential of these versatile reagents was first described by Cleland in 1964. Due to their low redox potential (—0.33 V) they are able to reduce virtually all accessible biological disulfides and maintain free thiols in solution despite the presence of oxygen. The compounds are fully water-soluble with very little of the offensive odor of the 2-mercaptoethanol they were meant to replace. Since Cleland s original report, literally thousands of references have cited the use of mainly DTT for the reduction of cystine and other forms of disulfides. 

Isolation of complexed molecules may be done by affinity chromatography using a column of immobilized avidin or immobilized streptavidin. Cleavage of the disulfide bond of the crosslinker may be done by treatment with 50 mM dithiothreitol (DTT). For additional information on the use of sulfo-SBED in the study of protein interactions, see Chapter 28, Section 3.1. 

The following protocol for labeling proteins with 5-IAF is adapted from Gorman (1987). It is a bit unusual in that it involves reduction of disulfides with dithiothreitol (DTT) and immediate reaction with 5-IAF in excess without removal of excess reductant. The procedure can be changed to include a gel filtration step after disulfide reduction to remove excess DTT, but in any case, it should be optimized for each protein to be modified. An alternative to the use of DTT to produce sulfhydryls is thiolation with a compound that can generate free thiols upon reaction with a protein (Chapter 1, Section 4.1). 

Wash particles (e.g., 100 mg of 1 pm carboxylated latex beads) into coupling buffer (i.e., 50 mM MES, pH 6.0 or 50 mM sodium phosphate, pH 7.2 buffers with pH values from pH 4.5 -7.5 may be used with success however, as the pH increases the reaction rate will decrease). Suspend the particles in 5 ml coupling buffer. The addition of a dilute detergent solution may be done to increase particle stability (e.g., final concentration of 0.01 percent sodium dodecyl sulfate (SDS)). Avoid the addition of any components containing carboxylates or amines (such as acetate, glycine, Tris, imidazole, etc.). Also, avoid the presence of thiols (e.g., dithiothreitol (DTT), 2-mercaptoethanol, etc.), as these will react with EDC and effectively inactivate it. 

Reduce protein disulfides by adding dithiothreitol (DTT) to a final concentration of 5mM and incubate for 30 minutes at 60°C. Add iodoacetamide to a final concentration of 25 mM to alkylate the thiols. React for 1 hour in the dark. 

Screening of over 66,000 compounds from the MLSMR by scientists at the PCMD for inhibitors of Cathepsin B resulted in the identification and characterization of an alternate substrate, SID 16952359 [29]. This study also describes issues relating to the nucleophilicity of dithiothreitol (DTT) and cysteine, reductants frequently used in HTS protocols, and the potential for reactivity with electrophilic sites of probe molecules. 

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