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M Saleemuddin

Publications and source records attributed to M Saleemuddin.

51 records · Page 3Linked to original sources
PubMed1987

Protein assay by Coomassie brilliant blue G-250-binding method is unsuitable for plant tissues rich in phenols and phenolases.

Protein estimation in crude homogenates of plant tissues rich in phenols and phenolases was carried out by the dye-binding and, with recommended cautions, by the Lowry et al. methods and the two were compared. The dye-binding method gave grossly erroneous results with a high degree of variation when the homogenizing media differed; this was not due either to the interference by the components of the homogenizing media or to any shift in the absorbance maximum. While the reduced form of the "derived" polyphenolic compounds, generated during tissue homogenization, appeared to enhance dye binding with bovine serum albumin, their influence on the protein assay directly in crude homogenates was extremely diverse. Tissue homogenization in the absence of a reducing agent results in polyquinone-protein complexes which prevent optimal dye binding, resulting in low protein values, while the endogenous phenolics in a homogenate prepared in a mixture of cysteine and NaCl appear to suppress dye-protein complex formation. It is therefore our opinion that the dye-binding method is unsuitable for protein assay in phenol- and phenolase-rich plant tissues.

Catechol Oxidase↗
PubMed1985

A Coomassie blue-binding assay for the microquantitation of immobilized proteins.

A sensitive assay procedure for the determination of microgram quantities of immobilized proteins is described. The procedure is based on the property of Coomassie blue G-250 to bind strongly yet reversibly to proteins. The assay involves incubation of the immobilized protein with a solution containing 0.1% Coomassie blue, 10% acetic acid, and 25% isopropyl alcohol in distilled water at room temperature followed by washing off of the unbound dye. The protein-bound dye is eluted with methanolic NaOH, acidified, and the absorbance is measured at 605 nm. The assay is highly reproducible and several proteins immobilized on various matrices could be conveniently assayed. Protein values determined by the dye-binding assay showed good agreement with those obtained by other procedures.

Amines↗
PubMed1983

Bromophenol blue protein assay: improvement in buffer tolerance and adaptation for the measurement of proteolytic activity.

A modification of the bromophenol blue dye binding procedure of protein estimation is described. Substitution of glycine/phosphoric acid, pH 2.6, for dilute acetic acid in the colour reagent extended the applicability of the procedure to protein solutions containing buffers of various pH values. This was, however, accompanied by approximately 25% loss in the sensitivity of the procedure. The modified reagent exhibited very marked tolerance to detergents and could be successfully adapted for the measurement of proteolytic activity in acidic, neutral or alkaline pH ranges.

Bromphenol Blue↗
PubMed1978

Use of glyceraldehyde-3-phosphate dehydrogenase-depleted human erythrocyte ghosts as specific high affinity adsorbents for the purification of glyceraldehyde-3-phosphate dehydrogenase from various tissues.

Human erythrocyte ghosts depleted of glyceraldehyde-3-phosphate dehydrogenase are used as specific high-affinity adsorbents for the purification of glyceraldehyde-3-phosphate dehydrogenase from mouse muscle, liver, kidney and brain. On incubation with the crude tissue homogenates, the depleted ghosts bind glyceraldehyde-3-phosphate dehydrogenase, aldolase, and a few other proteins. Washing the incubated ghosts several times with 5 mM phosphate buffer(pH 8.0) removed several of the non specifically bound proteins. Aldolase can be eliminated from the membrane by incubating the ghosts for 30 min in 5 mM phosphate buffer (pH 8.0)/2mM fructose 1,6-biphosphate, and then washing with the same solution. Glyceraldehyde-3-phosphate dehydrogenase can then be specifically eluted from the ghosts by incubating them with 2 mM NADH in 5mM phosphate buffer (pH 8.0). Although the enzyme from brain appears to bind less strongly to the ghosts it was possible, using this procedure, to purify glyceraldehyde-3-phosphate dehydrogenase from all the tissues investigated. The purified enzyme exhibits high specific activity and migrates as a single band (during SDS polyacrylamide gel electrophoresis) which corresponds to a protomer molecular weight of 37 000.

Animals↗
PubMed1977

Preparation of uniform haemoglobin free human erythrocyte ghosts in isotonic solution.

A method is described for the preparation of haemoglobin free human erythrocyte ghosts in isotonic solutions using dielectric breakdown technique. In this single haemolytic procedure, almost complete removal of haemoglobin (less than or equal to 0.1%) was achieved by subjecting the erythrocytes suspended in phosphate buffered, isotonic KCl solution at 0 degrees C to three consecutive electrical field pulses of 16 kV/cm in the presence of 10 mM EDTA; EDTA was used to prevent electrical haemolysis. Haemolysis is induced by subsequent dilution with isotonic and isoionic solution to lower the EDTA concentration. Haemolysis is complete after 5 min; the cells are centrifuged, washed and resuspended in a solution of the same composition and osmolarity containing 4 mM MgCl2, but no EDTA. The resealing process, carried out at 37 degrees C, was complete in about 1 h. Measurements of the size distribution of the ghost cells in the hydrodynamically focusing Coulter Counter at varying field strengths in the orifice revealed that the ghost population is nearly uniform. The mean (modal) volume of the ghost cells was 110--120 micronm3 when suspended in phosphate buffered NaCl solution. The apparent breakdown voltage was about 1.3 V.

Erythrocyte Membrane↗
PubMedDate not supplied

Preparation of human erythrocyte ghosts in isotonic solution: haemoglobin content and polypeptide composition.

Human red blood cell ghosts were prepared by dielectric breakdown of the cell membrane and subsequent electrical haemolysis in isotonic and isoionic solutions. Almost complete removal of haemoglobin (to about 0.1%) could be achieved by subjecting the erythrocytes suspended in isotonic solution to three consecutive electric field pulses of 16 kV/cm in the presence of 10 mM EDTA, followed by dilution with EDTA-free isoionic medium. The cup-shaped ghost cells were electrically homogeneous. The polypeptide composition of the electrically prepared ghost cells, as analyzed by polyacrylamide electrophoresis in 1% SDS, revealed a pattern similar to ghost cells prepared by osmotic haemolysis. However, the band 6, corresponding to glyceraldehyde-3-phosphate-dehydrogenase protomer, was absent from the electrically prepared ghost cells. Also the band 8, which is eluted at low ionic strength, appeared to be very prominent in the electrical preparations.

Cell Fractionation↗