Protein estimation assay

Membrane protein content can be determined using a protein estimation assay (e.g., Pierce BCA assay). The membrane protein content is necessary to estimate receptor expression levels (Bmax) from binding data. 

Plus Protein Assay Reagent was used, the total protein (IgG) concentration in the sample would be underestimated by -40%. (From Table B1.1. 5, the response ratio for IgG is -0.58 for IgG compared to 1.00 for BSA.) If the BCA Protein Assay Reagent was used, the total protein (IgG) concentration in the sample would be overestimated by -15%. (From Table B1.1.5, the response ratio for IgG is -1.15 for IgG compared to 1.00 for BSA.) On the other hand, if BGG had been used for both standard curves, the total protein estimates for the sample would have been in much greater agreement between the two methods. 

Some important reactions are those which are used to estimate the concentration of proteins. These assays all assume that the reacting groups of a protein have an average distribution through the protein molecule. 

Artefactual increases of as much as 50% in total thyroxine, estimated by a competitive protein-binding assay, and of as much as 30% in triiodothyronine resin uptake are probably due to rapid and continuing lipolytic hydrolysis of triglycerides after blood has been drawn (126). Thyroid function tests should therefore always be performed on blood samples taken before (or a sufficient time after) heparin treatment (127). An increase in serum-free thyroxine concentrations has also been reported after low molecular weight heparin, by up to 171% in specimens taken 2-6 hours after injection. When specimens were obtained 10 hours after injection, the effects were smaller, but with concentrations still up to 40% above normal the results can still cause errors of interpretation (128). 

Figure 1.5. Relationship between actual and measured BSA concentration in six samples, showing that this method produces comparable results to the Bradford and BCA total protein assays. [Reprinted, with permission, from K. C. Bible, S. A. Boemer, and S. H. Kaufmann, Anal. Biochem. 267, 1999, 217-221. A One-Step Method for Protein Estimation in Biological Samples Nitration of Tyrosine in Nitric Acid. Copyright 1999 by Academic Press.]...

A3. Archibald, E. L., Mincey, E. K., and Morrison, R. T., Estimation of serum folate levels by competitive protein binding assay. Clin. Biochem. (Ottawa) 5, 232-241 (1972). 

Use the correct Pierce BCA kit (Pierce Biotechnology Cat. No. 23225 or 23227) for determination of the protein concentration the other kits will be affected by the components of our buffers. Pierce s BCA Protein Assay Reagent Kit should be used for all Ab microarray analyses. Using other BCA reagents (or kits) could lead to errors in protein estimation, because Ab microarray buffers contain substances known to interfere with protein assays. Pierce s Kit has been tested by our scientists and approved for use with Ab microarray procedures and reagents. 

We recommend you use the BCA Protein Assay Reagent Kit from Pierce Biotechnology. It has been tested by our scientists and shown to be compatible with our buffers. Using other BCA reagents (or kits) could lead to errors in protein estimation. 

Final concentration can be estimated by comparison to ESA standards by SDS-PAGE analysis or Bradford protein determination assays... 

Transcortin can be used in competitive protein binding assays for the estimation of cortisol. 

Latex agglutination immunoassays are easily formatted into simple kits which can provide yes/no and semiquantitative estimates of antigen (or antibody) in a sample. The first such assay was developed in 1957 for rheumatoid factor (15) and assays are on the market for the deterrnination of many species of bacteria, fungi. Mycoplasma, parasites, ckettsia, and vimses, as well as for the deterrnination of autoimmune disease, hormones (qv), dmgs (see Pharmaceuticals), and blood proteins (16). Latex agglutination is also the basis of many home pregnancy tests. 

The quality control of immunoglobuhns includes potency tests and conventional tests of safety and sterihty. The potency tests consist of neutrahzation tests that parallel those used for the potency assay of immunosera, except that in the cases of some immimoglobulins the assays are made in vitro, fit addition to the safety and sterihty tests, total protein is determined by nitrogen estimations, the protein composition by... 

Hapten density is important for both immunization and assay performance, and hence the extent of conjugation or hapten density should be confirmed by established methods. A characteristic ultraviolet (UV) or visible absorbance spectrum that distinguishes the hapten from the carrier protein or use of a radiolabeled hapten can be used to determine the degree of conjugation. If the hapten has a similar A. iax to the protein, the extent of incorporation can still be estimated when the concentration of the protein and the spectral characteristics of the hapten and protein are known. The difference in absorbance between the conjugate and the starting protein is proportional to... 

Here m is the slope value and [ii]app is the apparent total enzyme concentration, typically estimated from protein assays and other methods (Copeland, 1994). Note from Equation (7.13) that when our estimate of enzyme concentration is incorrect, the slope of the best fit line of IC50 as a function of [E] will not be 1/2, as theoretically expected. Nevertheless, the v-intercept estimate of K pp is unaffected by inaccuracies in [ ]. In fact we can combine Equations (7.12) and (7.13) to provide an accurate determination of [ /]T from the slope of plots such as those shown in Figure 7.2. The true value of [ii]T is related to the apparent value [ TPP as... 

Add iodoacetate to a concentration of 50mM in the reaction solution. Alternatively, add a quantity of iodoacetate representing a 10-fold molar excess relative to the number of —SH groups present. An estimation of the sulfhydryl content in the protein to be modified can be accomplished by performing an Ellman s assay (Chapter 1, Section 4.1). Readjust the pH if necessary. To aid in adding a small quantity of iodoacetic acid to the reaction, a concentrated stock solution may be made in the reaction buffer, the pH re-adjusted, and an aliquot added to the protein solution to give the desired concentration. 

Citrated blood is diluted 1 10 with enzyme buffer solution, and preservative is added (H19). The buffer is prepared by dissolving 0.2 g of Clarase (Fisher Scientific Co., New York) in 100 ml citrate buffer (5 g potassium citrate monohydrate and 1 g citric acid monohydrate in 1000 ml distilled water, pH 5.6). The solution is incubated for 3 days at 37°. After incubation, it is autoclaved 15 minutes to stop enzymatic action and coagulate proteins. It is filtered, and 1.0, 1.5, and 2.0 ml of the supernatant is added to individual flasks and assayed. Control flasks are included to estimate pantothenic acid contamination of the enzyme. 

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