Ninhydrin stain

Fig. 4.2 Hydrolytic activity of cauxin on 3-MBCG and proposed metabolic pathways for the conversion of 3-MBG to felinine in the cat kidney. The 3-MBCG (20 mM) was incubated with or without cauxin (1.5 mg/ml) at 38°C for 6 h, and the reaction mixtures were analyzed by thin layer chromatography with ninhydrin staining. y-GTP, y-glutamyl transferase RDP, renal dipeptidase a, 3-mercapto-3-methylbutyl formate b, 3-mercapto-3-methyl-l-butanol c, 3-methyl-3-methylthio-1-butanol and d, 3-methyl-3-(2-methyldisulfanyl)-l-butanol...

CAUTION Methyl cellosolve is toxic and ninhydrin stains everything it contacts. 

A variety of methods has been used in the study and screening of inherited enzyme defects. Partition chromatography on paper with ninhydrin staining for identifying amino acids [338], TLC and more recently gas chromatography, e.g. [339-341] have all been employed extensively. However the positive characterisation of compounds by GC-MS has been a very powerful additional technique. It has been used in... 

B12. Brummer, F., and Kulonen, M., Chromatographic pattern of ninhydrin staining compounds in gastric juice and its relation to acid gastric secretion. Acta Med. Scand, 167, 61-64 (1960). 

The freeze-dried peptide is hydrolyzed in 6 N HCl at 110°C for 1 h, HCl is removed by evaporation, and the residue is dissolved in H2O, mixed with unlabeled phosphoamino acid markers, and electrophoresed on the Kodak cellulose sheets (Erdodi et al., 1987). The phosphoamino acids are identified by ninhydrin staining and autoradiography (Rg. 2). The procedure is only qualitative, because of the incomplete hydrolysis of the phosphopeptide and the partial destruction of the liberated phosphoserine and phosphothreonine. 

The formation of the imines and their subsequent reduction was monitored by the disappearance of the starting amine (ninhydrin staining, TLC). Simple quenching with water and drying of the organic solutions with MgS04 constituted the reaction work-up. The condensation of the amines with isothiocyanates and TEA was monitored by TLC (blue bromophenol staining, disappearance of... 

Ninhydrin staining solution Dissolve 0.25 g ninhydrin in 100 ml acetone. 

Spray the plate with 0.25% ninhydrin in acetone and bake 15 min at 65°C to develop the stain of the three phosphoamino acid markers. Ninhydrin stains primary amino groups and the phosphoamino acid standards appear as purple spots (see Fig. 5 for key to the positions of the standards). The staining persists during autoradiography. 

Keller et al. (1984). separated eross-linking amino acids of elastin by two-dimensional silica gel TLC using butanol-acetic acid-water (4 1 1) and propa-nol-NH,-water (8 1 11). Schwartz (1984) detected as little as 60 pmol of histidine phenylthiohydantoin by UV (366 nm) irradiation on a fluorescein-containing silica gel plate after development with ethanol-acetic acid (7 3). Henderson et al. (1985) reported that two-dimensional TLC was useful for screening aspartyl-glycosaminuria in the urine of children by heating ninhydrin-stained plates at 120°C. 

Matrix-assisted laser desorption ionization (M ALDI) has also been used for TLC/MS by Gusev et al. (61). Absolute detection limits of 2-4 ng were demonstrated for bradykinin, angiotensin, and enkephalin derivatives. Application of the MALDI support matrix to the TLC plate after separation is completed induces a planar diffusion of 1-1.5 mm. In an interesting application of the MALDI TLC/MS method, the mass spectra of ninhydrin-stained spots were obtained. Ions corresponding to the ninhydrin adducts with the sample molecules could be observed. A spatially resolved image for bradykinin on a TLC plate in a 2-(4-hydroxyphenylazo) benzoic acid matrix was also reported in this paper. 

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