Showing papers on "Bradford protein assay published in 1995"

A Novel Protein Assay Method Using Tetraphenylporphin tetrasulfonate (TPPS4)

Abstract: A sensitive protein assay method which involves the reaction of TPPS4 with protein is described. When protein is added to TPPS4 solution, an absorption band with the maximum at 488 nm appears and the absorbance is proportional to the concentration of protein. Just Like the Soret absorption of the porphyrin, the new band is very narrow and there is no overlap at all between them, which means the free dyes would not give any background for the detection of the protein-TPPS4 complexes. A new spectrophotometric method for determination of protein has been constructed and applied to the determination of human plasma protein and urinary protein; The assay using microtiter plates has also been studied.

Protein concentration by precipitation with pyrogallol red prior to electrophoresis.

Abstract: The pyrogallol red protein assay (Clinical Chemistry 1986, 32, 1551-1554) is based upon formation of a blue protein-dye complex in the presence of molybdate under acidic conditions. However, centrifugation of the assay mixture results in loss of color yield and precipitation of the protein-dye complex which can be recovered and resolubilized to achieve protein concentration prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The method has been evaluated relative to trichloroacetic acid (TCA) precipitation for recovery and electrophoresis of commercial protein and peptide molecular weight markers. Precipitation with pyrogallol red-molybdate (PRM) gives better and more uniform recovery of both proteins and peptides as compared to TCA. The lower limit of PRM precipitation is similar to TCA and corresponds to 1 microgram protein per mL assay mixture. This is equivalent to 100 microL of 10 micrograms/mL protein using the standard protein assay or 1 microgram/mL protein using a modified assay incorporating a fivefold concentrate of the dye reagent. Application of the method is demonstrated by concentration of urinary proteins. The method is simple and economic and useful for conserving trace amounts of precious sample as it allows recovery of protein for electrophoresis following protein assay.

Determination of surface hydrophobicity of milk proteins. Comparison between a new detergent binding method and hydrophobic interaction-FPLC

Abstract: A previously published method for the determination of the surface hydrophobicity of proteins using BIO-RAD protein assay combined with Tween 80 was adopted to various milk proteins. The relative retention times obtained by HI-FPLC at Phenyl-Superose for casein and whey protein were compared with results of the proposed method and expanded by the average hydrophobicity calculated according to BIGELOW. Detergent binding showed good correlation with calculated average hydrophobicities for all milk proteins, whereas HI-FPLC results were found to be accurate only for caseins. The relative order of hydrophobicity for single caseins was pH dependent. In contrast, HI-FPLC was found to be not selective for separation of whey proteins according to their hydrophobicity, but selective for the solubility characteristics related from the used buffer system. Evidence is given that this proposed method can be easily performed, is highly reliable and adaptable to a rapid quantitative differentiation between hydrophobic and hydrophilic areas on protein surfaces

Urinary Protein and Albumin Determinations by High-Performance Gel-Permeation Chromatography

Abstract: Sensitive high-performance gel-permeation chromatographic protein assay method was developed by using non-ionic detergent of Brij-58 which was UV transparent as compared to Nonidet P-40. A column (70 × 8.0 mm I.D.) packed with Develosil 100 Diol-5 (pore size 10 nm) was used for the protein determination. A commercially available column (300 × 8.0 mm I.D.) packed with Develosil 300 Diol-5 (pore size 30 nm) was used for the urinary albumin determination. Eluent used was a 0.1 M sodium phosphate buffer (pH 5.6) containing 0.3 M sodium chloride, 1% (v/v) Brij-58, 50% (v/v) glycerol. Flow-rates for protein assay and albumin assay were 1.0 and 0.5 ml/min, repectively. Proteins were eluted at the position of the exclusion limit in the case of protein assay (100 Diol-5). Albumin was eluted from 300 Diol-5 column as a symmetric peak. Bovine serum albumin (BSA) was used as an external standard for both protein and albumin assays. Analysis times were within four min for protein assay, and 32 min for albumin...