Glutaraldehyde: nature of the reagent

Aqueous solutions of glutaraldehyde used for the chemical modification and stabilization of proteins have been found to consist of free glutaraldehyde (I), the cyclic hemiacetal of its hydrate (II) and oligomers of this (III) in equilibrium with each other. Ultraviolet absorption spectra of these solutions at wavelengths greater than 200 nm should have only a single maximum at 280 nm. Absorption at 235 nm is due to an impurity which can be removed by various means. Reactions of the reagent with proteins involve principally the lysinyl (and hydroxylysinyl) residues in the relative amounts of four moles of glutaraldehyde to one of lysine. Three unstable products can be partially isolated from acid hydrolyzates of glutaraldehyde-treated proteins or from the reaction mixtures of glutaraldehyde and model compounds; two of these products have strong ultraviolet absorption near 265 nm.     

An alcohol-soluble Schiff's reagent: A histochemical application of the complex between Schiff's reagent and phosphotungstic acid

Summary A schedule for staining partially hydrated PAS-positive structures using non-aqueous solutions has been devised. Tissues are dewaxed, taken down to 70% alcohol, oxidised for 10 min in a 1% w/v alcoholic solution of periodic acid, treated with an alcoholic solution of phosphotungstic acid-Schiff reagent complex (PTA-Schiff reagent) for 25 min, washed in alcohol, cleared in xylene and mounted in a synthetic medium. The PTA-Schiff reagent complex prepared from de Tomasi Schiff reagent by precipitation with PTA may be stored in the deep freeze for many months and dissolved freshly in alcohol for use. The PTA-Schiff reagent sued as above allows staining of highly water soluble materials such as dextran. From blocking and digestion studies the mode of action seems similar to de Tomasi Schiff reagent. The partial hydration of the tissues prior to reaction was found to be essential for effective staining.     

An alcohol-soluble Schiff's reagent: a histochemical application of the complex between Schiff's reagent and phosphotungstic acid.

A schedule for staining partially hydrated PAS-positive structures using non-aqueous solutions has been devised. Tissues are dewaxed, taken down to 70% alcohol, oxidised for 10 min in a 1% w/v alcoholic solution of periodic acid, treated with an alcoholic solution of phosphotungstic acid-Schiff reagent complex (PTA-Schiff reagent) for 25 min, washed in alcohol, cleared in xylene and mounted in a synthetic medium. The PTA-Schiff reagent complex prepared from de Tomasi Schiff reagent by precipitation with PTA may be stored in the deep freeze for many months and dissolved freshly in alcohol for use. The PTA-Schiff reagent used as above allows staining of highly water soluble materials such as dextran. From blocking and digestion studies the mode of action seems similar to de Tomasi Schiff reagent. The partial hydration of the tissues prior to reaction was found to be essential for effective staining.     

Determination of proteins and sulfobetaine with the folin-phenol reagent

This paper describes a method for the quantitative analysis of solutions containing a mixture of proteins and sulfobetaine. In a preliminary step the proteins, which interfere with the detergent assay, are separated by precipitation with trichloroacetic acid (8%). The insoluble fraction, dissolved in NaOH (1.0 n), and the soluble fraction, containing the detergent, are treated with the Folin-Ciocalteu phenol reagent, essentially following the method of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951, J. Biol. Chem.193, 265–275). The absorbance of the protein fraction is read, as usual at 750 nm, while that of the detergent solution is read at 342 nm. At this wavelength, sulfobetaine, treated with the Folin reagent, absorbs strongly, the absorbance being proportional to its concentration up to 1.5 mg/ml.     

A glutaraldehyde-fuchsin reagent

Synopsis A glutaraldehyde-fuchsin reagent is described. Its preparation avoids the time-consuming ripening period, associated with Gomori's aldehyde-fuchsin. Elastic tissue, insular -cells (after an appropriate prior oxidation) and various other tissue components (e.g. pituitary basophils and acid mucosubstances) can be stained with the reagent 1–2 hours after its preparation.     

A reagent forum

My lab works with the yeast Saccharomyces cerevisiae, but I frequently get asked questions regarding autophagy reagents for use in higher eukaryotic systems. For example, "Which anti-LC3 antiserum works well in HEK293 cells"? Occasionally I know the answer, or an answer, but not always. Sometimes the information is available in the literature, but it can be difficult to find. With the ever-growing community of autophagy researchers, however, I suspect that many more reagents have been tested, and in many cases not reported. Therefore, I think it would be useful to establish a list of reagents (in particular, antisera and inhibitors) with company-specific information (name and catalog number) that can save other researchers valuable time and money. Autophagy will post the information in the form of a WIKI to allow updates and comments from users.

For example:

Reagent: anti-LC3

Company: Count On Us

Catalog number: 01234

Comments:

Dan Klionsky: I used this antibody and had a very good result (good sensitivity with low background) on protein extracts from mouse.

Danny Klionsky: I also had a good result with this antibody, and found that a higher dilution (1:3,000) worked just as well with rat liver protein extract.

Daniel J. Klionsky: This antibody did not work for yeast Atg8.

We plan to start the WIKI in August 2008, but its success depends entirely on your participation. Please check for the start of this site, and upload your entries. I am open to suggestions for improvements of the site that will make it more useful for autophagy researchers.

http://www.landesbioscience.com/reagent_blog/     

A new reagent for the preparation of glycoconjugates

The thioglycoside derivative 2-carbazoylethyl 1-thio-beta-D-galactopyranoside hydrochloride was synthesized. Conversion of the carbazoyl group into the reactive azidocarbonyl function leads to a well suited reagent for the preparation of glycoconjugates via amidation of proteins or synthetic carriers in aqueous media.     

Folin试剂制备中的一个改进

本文介绍了Folin试剂制备中的一个改进,使制备过程得到简化.