Bicinchoninic acid methods

Total protein is measured using the bicinchoninic acid method (BCA assay Pierce Co., Rockford, IL). 

Bicinchoninic acid method Bicinchoninic acid is added to the reaction products of the biuret reaction to form the copper-bicinchonate complex. The absorbance of the complex is measured spectrometrically at 560 nm 0.5-50 ng ml" ... 

Protein concentration in the fractions were determined using the bicinchoninic acid method (Pierce) with BSA as standard. 

This is a modification of the Lowry method involving a dye-binding step. The copper-protein complex that forms the basis of the biuret and the Lowry methods can be chelated by bicinchoninic acid to produce a very stable complex with a strong absorption maximum at 562 nm. 

This unit describes four of the most commonly used total protein assay methods. Three of the four are copper-based assays to quantitate total protein the Lowry method (see Basic Protocol 1 and Alternate Protocols 1 and 2), the bicinchoninic acid assay (BCA see Basic Protocol 2 and Alternate Protocols 3 and 4), and the biuret method (see Basic Protocol 3 and Alternate Protocol 5). The fourth is the Coomassie dye binding or Bradford assay (see Basic Protocol 4 and Alternate Protocols 6 and 7), which is included as a simple and sensitive assay, although it sometimes gives a variable response depending on how well or how poorly the protein binds the dye in acidic pH. A protein assay method should be chosen based on the sensitivity and accuracy of method as well as the condition of the sample to be analyzed. 

Monitoring of solubility and dissolution Gravimetric analysis, a protein assay method (involving color reaction with bicinchoninic acid) and UV absorbance measurements at 214 nm Chemical analysis using TNBS reagent Fluorescence spectrophotometry... 

The three most common assays for total protein2 are the Lowry (enhanced copper), Smith (bicinchoninic acid, BCA), and Bradford (Coomassie Blue) methods. All are colorimetric methods, and are based on the generation of absorbing species in proportion to the quantity of protein present in the sample. The ninhydrin assay is a recently reported promising method. 

Method Biuret Reaction Bicinchoninic Acid Lowry Bradford... 

Colorimetric assays are appropriate methods to investigate protein interactions with cationic polysaccharide materials. However, these assays cannot differentiate between different types of interactions, i.e. adsorption and absorption of proteins. Among these, the bicinchoninic acid assay (BCA) is widely used since it allows an easy determination of the total amount of adsorbed proteins on bulk materials." The assay is based on the reduction of Cu to Cu in a reductive environment in the presence of proteins. The BCA chelates Cu, forming a colored complex whose absorption band at 562 nm can be related to the amount of adsorbed proteins on the materials. Using this method, it has been demonstrated that the incorporation of inorganic materials such as hydroxyapatite (HAP) or nano-HAP into chitosan-based materials leads to the formation of protein-rich layers at the... 

Use either the Bradford or bicinchoninic acid (BCA) method to quantify total protein concentrations following the manufacturer s instructions for a microweU plate assay. Make sure that the lysis buffer components are compatible with the manufacturer s stated levels. Try several dilutions to ensure the sample concentration is within the linear range of the assay. 

The most common of these assays are the Bradford, Lowry, and bicinchoninic acid (BCA) methods. In addition to these classical absorbance-based methods, convenient fluorescence-based methods are becoming increasingly more popu-lar. Some salient aspects of these assays are summarized in Table 3. 

Alkaline phosphatase (ALP) activity ALP activity of the supematarrt was meastrred using an ALP B-Test WAKO kit (Wako, Japan) based on the Bessey-Lowry method. ALP activity was normalized by samples total protein production measured with a Bicinchoninic Acid Assay (BCA, BCA Protein Assay KiL Pierce Biotechnology, U.S. A.). 

Collect the supernatant and determine the protein concentration using the Bradford reagent or an alternative method for determination of total protein (e.g., Lowry, Bicinchoninic Acid (BCA)). 

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