Method For Quantitation Of Proteins Research Articles

Rapid method for quantitation of androgen binding protein in sertoli cell cultures and its use for measurement of binding kinetics

The accurate measurement of the kinetics of binding of 5α-dihydrotestosterone to the Sertoli cell specific protein, androgen binding protein (ABP), has been frustrated by the extremely rapid rate of dissociation of the ABP-dihydrotestosterone complex. We describe a rapid and highly sensitive assay suitable for ABP quantitation which utilizes DEAE Bio-Gel and [3H]dihydrotestosterone. The assay has been used to accurately measure the rate of dissociation (8.25 × 10−4s−1, t12 14min) and the rate of association (2.04 × 105M s−1) of the binding of [3H]dihydrotestosterone to rat ABP. The ratio of these rate constants is in perfect agreement with the equilibrium dissociation constant determined by Scatchard analysis (4.0 nM). This multipoint assay is extremely rapid such that binding can be measured at equilibrium, it has high precision (coefficient of variation 3%), and is particularly useful at low protein concentrations (50 ng/ml); furthermore, the assay background of nonspecific 3H-binding is extremely low (0.2%). Since at such low protein concentrations a 10 point Scatchard analysis can be performed on 1 ml culture medium containing as little as 3 fmol ABP, the assay is suitable for monitoring changes in ABP secretion resulting from manipulations of cells in culture. The assay which utilizes DEAE Bio-Gel A is compared to five alternative methods: the standard method of steady state gel electrophoresis, Dextrancoated charcoal assay, hydroxylapatite assay, DEAE filter assay, and radioimmunoassay. The DEAE Bio-Gel assay has advantages over all of these alternative methods. In summary, this new assay is particularly useful for monitoring temporal changes in the secretion of ABP, and the method is equally effective in quantitating ABP in rat, rabbit and hamster Sertoli cell cultures.

A method for isolation, characterization, and quantitation of soluble proteins in uterine cervical secretion.

By sodium chloride (0.66 M) extraction of cervical mucus on an inert chromatographic bed, soluble proteins can be conveniently recovered. Individual proteins such as albumin and IgG may then be quantitated by immunonephelometry. The procedure has been shown to improve reproducibility which is very poor in the presence of mucin. By concentrating the NaCl extract by centrifiltration, proteins present in low concentrations - such as complement C3 - can be measured. The concentrate has also been used for analyses by isoelectric focusing or gradient gel electrophoresis on polyacrylamide gels.

A simple, sensitive enzymatic method for quantitation of soya proteins in soya-meat blends

A method was developed for the detection and quantitation of soya proteins in soya-meat blends. The procedure involves acid hydrolysis of soya, meat or soya-meat blends and determination of free galactose plus arabinose using galactose dehydrogenase. Acetone powders of the various systems were also examined. Average galactose levels in soya flours, soya concentrates and soya isolates were 674,600 and 66 μM galactose equivalents per gram of product, respectively. Beef and pork contained less than 1 μM per gram of raw product. The galactose plus arabinose values for soya-meat blends were linearly dependent on the amount of soya added. The method is sensitive and capable of detecting less than 0·2% soya flours or concentrates and less than 2% soya isolates in meat products.

A novel method for protein quantitation using a competitive binding assay

A method for determining protein concentration in the range of 5 × 10 −7 to 1 × 10 −4 g protein/ml is described. A polypropylene solid phase is activated to bind proteins by treatment with a glutaraldehyde solution. A competition is then set up between a protein of unknown concentration, and a known quantity of alkaline phosphatase for the reactive sites on the solid phase. After protein binding to the solid phase has occurred, enzyme activity on the solid phase is determined. Enzyme activity varies inversely with the concentration of the competing protein.

A method for quantitation of protein in the presence of percoll

A simple assay for protein in the presence of Percoll which requires neither additional instrumentation nor manipulation of the sample is proposed. The method is based on monitoring the absorbance at 750 and 420 nm of color developed by protein plus Percoll with Folin-Ciocalteu reagent. Protein can be determined in a sample containing as much as 30% Percoll. The method does not require the knowledge of Percoll concentration (below 30%) in the sample. A large number of samples from a Percoll density gradient can be assayed very conveniently and with great precision.

Protein transfer to nitrocellulose filters: A simple method for quantitation of single proteins in complex mistures

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Refinement of the Coomassie blue method of protein quantitation: A simple and linear spectrophotometric assay for ≤0.5 to 50 μg of protein

The Coomassie brilliant blue G assay for proteins described by Bradford (1976) ( Anal. Biochem. 72, 248) was reexamined. It was found that the extinction coefficient of the dye-protein complex solution remained constant over the protein concentration range of 0.8 to 10 μg/ml of solution. This unchanging extinction coefficient, A 595 = 0.60 ± 0.01 10 μ g of protein/ml of solution, enhances both the sensitivity and versatility of the assay. Selection of a volume of dye-reagent (0.5 to 5.0 ml) which dilutes the protein sample to a final concentration of 0.8 to 10 μg/ml permits the application of Beer's Law for accurate determinations of ≤0.5 to 50 μg of protein. A combination of Bradford's study and the present one indicates that most common laboratory reagents and chemicals exert little or no influence on the A 595 of the dye-reagent.

Quantitation of microgram amounts of protein in SDS-mercaptoethanol-tris electrophoresis sample buffer

We have investigated the utilization of a previously published dye-binding protein quantitation method for the analysis of proteins solubilized in SDS, mercaptoethanol-tris electrophoresis sample buffer. Although there is a direct relationship between the amount of protein applied and the absorbance of Coomassie blue G for BSA standard curves, complicated protein mixtures such as whole cell homogenates of tissue culture cells show more complicated functions. Apparently, components present in whole cell homogenates altered the dye-binding phenomenon to the extent that absolute values of the total amount of protein of such a mixture cannot be obtained by comparison to standard curve. However, over a relatively wide range of protein concentrations, the dye-binding method can provide a relative value for the amount of total protein present in a given complex protein mixture solubilized in SDS-mercaptoethanol electrophoresis buffer.

Molecular heterogeneity effects of immunoglobulin classes in radial immunodiffusion

Mancini et al. (1965) have reported a method for immunological quantitation of proteins by means of single radial diffusion, which is now used extensively in immunochemistry for measurement of immunoglobulin concentrations in sera. Using this method, in instances of monospecific antibodies, to quantitate immunoglobulins leads in some cases to difficulties because of multiple ring precipitations. In this communication we report multiple precipitation in single radial diffusion, using recently commercially available plates (Tri-partigen plates, Behringwerke, Marburg, Germany), as an indication of molecular heterogeneity in different immunoglobulin classes.

Rapid Method for Protein Quantitation by Electroimmunodiffusion

Abstract In the electroimmunodiffusion method described, a Mylar-backed cellulose acetate membrane plate is used as a support medium. Results are obtained faster, less preparation time is required, and the method is less expensive because less antisera is needed. Results with the method are reproducible (CV, 5%), correlate well with results by the single radial immunodiffusion method, and sensitivity is similar to the electroimmunodiffusion method in which agar gel is used as a support medium.

Quantitation of immunoglobulin G by electrophoresis in agarose gel containing antibodies.

It is shown that the method of protein quantitation by electrophoresis in antibody-containing agarose introduced by Laurell in 1966 can, if slightly modified, be used for quantitation of polyclonal human IgG. This modified method, permits simultaneous quantitation of IgA. It is accomplished within about 9 hours and allows comparison between the charge composition of different IgG-populations. The results agree with those obtained by single linear immunodiffusion, single radial immunodiffusion, and by electrophoresis of carbamylated sera in antibody-containing gel. It is shown that the applicability of the method is dependent upon the electrophoretic distribution of the antibodies in the antiserum used.

Determination of Total Serum Protein by Means of the Refractive Index of Serum

The most frequently used method for quantitation of serum protein in hospital laboratories is the biuret method. Utilization of the physical property of refractive index of a fluid to quantitate its contents is common in industry. A simple instrument, the refractometer, is used. By means of applying this theory in human serum,' 6 one can obtain accurate and rapid estimates of the content of total serum protein without the use of chemicals and glassware.