Ellman’s reagent

For quantitative analysis of solid supported thiol residues on free macroporous or PEG grafts, Ellman s reagent has been used [5,5 -dithio-hfr-(2-nitrobenzoic acid]. However only qualitative information can be gained using lightly crosslinked polystyrene resins [Badyal et al. Tetrahedron Lett 42 8531 2007]. 

The glutathione (GSH) level was assayed by the method of Sedlak and Lindsay (ref. 14), using Ellman s reagent (5,5-dithio-bis-(2-nitrobenzoic acid). Calibration was carried out parallely to each series, using reduced glutathione as a standard. 

Cysteine Iodoacetamide, maleimides, Ellman s reagent, p-hydroxymercuribenzoate... 

Ellman s Reagent Sulfhydryl Disulfide Bond 5-Thio-2-nitrobenzoic... 

Figure 1.79 The reaction of Ellman s reagent with a sulfhydryl group releases the chromogenic TNB anion, which can be quantified by its absorbance at 412nm.

Add 50 pi of Ellman s reagent to each standard and sample tube. Mix well. 

Mix the protein solution with an equal volume of the Ellman s reagent solution and react for 15 minutes at room temperature. 

Purify the modified protein from excess Ellman s reagent and reaction by-products by dialysis or gel filtration. A measurement of sulfhydryl content may be done by reading the absorbance of the modification reaction at 412nm (s = 1.36 X 104 M-1cm-1) versus a series of sulfhydryl standards treated in the same manner (e.g., cysteine). 

For one-step methods, the use of Ellman s reagent (previous section) to yield a similar reversible sulfhydryl blocking group is probably a better choice with protein molecules. 

After a carrier protein has been activated with sulfo-SMCC, it is often useful to measure the degree of maleimide incorporation prior to coupling an expensive hapten. Ellman s reagent may be used in an indirect method to assess the level of maleimide activity of sulfo-SMCC-activated proteins and other carriers. First, a sulfhydryl-containing compound such as 2-mercaptoethanol or cysteine is reacted in excess with the activated protein. The amount of unreacted sulfhydryls remaining in solution is then determined using the Ellman s reaction (Chapter 1, Section 4.1). Comparison of the response of the sample to a blank reaction using... 

Add 20 ul of Ellman s reagent (lmg/ml dissolved in the gel filtration purification buffer) to each well including one containing only buffer (220 pi) to use as a blank. 

Figure 21.11 Fab antibody fragments containing free thiols can be activated with Ellman s reagent to form a sulfhydryl-reactive derivative. A-chain toxin subunits containing a free thiol group may be coupled to the activated Fab molecule to produce an immunotoxin complex.

Riddles, P.W., Blakeley, R.L., and Zcrncr, B. (1979) Ellman s reagent 5,5 -dithiobis(2-nitrobenzoic acid)— A reexamination. Anal. Biochem. 94, 75-81. 

Ellman s reagent" + dithionite Synthetic vesicles of dicetyldimethyl-ammonium bromide. Effects of vesicles and fast and slow reaction components separated Moss and Schreck, 1983... 

Ellman s Reagent) [32]. Unfortunately, PLCBc processes this thiophospholipid 100 times slower than a natural phosphatidylcholine, so this assay is also of limited utility. 

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