The BCA Assay

Calculate the protein concentration in the final preparation using its absorbance at 280nm or a colorimetric method, such as the Coomassie assay. (Note The presence of hydrazine or hydrazide groups on the protein will interfere with the BCA assay for total protein concentration.)... 

Measure the absorbance of the biotinylated protein solution at 354 nm. Use the molar extinction coefficient for the chromogenic group (e = 29,000 M-1cm-1) to determine the concentration of biotin present. To determine the molar ratio of biotin-to-protein, divide the molar concentration of biotin by the molar concentration of protein present (which may be determined by using the Coomassie assay or the BCA assay methods). 

The BCA assay has a working range of 20 to 2000 Lig/ml. If the target protein is in a dilute aqueous formulation, the concentration can be determined with the micro BCA assay. The BCA protocol is modified by increasing the BCA concentration, incubation time, and incubation temperature. These modifications permit the detection of BSA at 0.5 Lig/ml. The major disadvantage of these modifications is that the presence of interfering substances decreases the signal-to-noise ratio and thus the sensitivity of the assay. 

The BCA assay for immobUized protein is more reliable than calculations from UV measurements of coupling supernatant. 

Since the BCA protein assay is not a true end-point assay, the amount of color produced varies with the incubation time and the incubation temperature. While this allows considerable flexibility in optimizing the BCA assay for each application, it also requires that the optimized procedure be followed exactly every time the assay is done. 

The reducing sugar-based assays are predicated on the correct measurement of enzymegenerated product. A typical calibration curve for the BCA assay, using galacturonic acid as the calibration standard, is presented in Figure Cl.2.5. Note that the assay is linear for the... 

Furthermore, every protein determination is sensitive to detergents or certain ions. Hence, when presenting a concentration value it is good practice to also mention the assay that was used as well as the benchmark protein. The methods of choice are the Bradford assay and the BCA (bicinchoninine acid) assay. Anyone working with membrane proteins and detergents should use the BCA assay. Otherwise, the choice between the BCA and the Bradford assays seems to be a question of taste. 

The eluate contains polybuffer/ampholines. This interferes with some assays (e.g., with the BCA assay see Section 1.2.1). You remove them using one of the following techniques. 

The Lowry and BCA methods are related, in that the first step of the assay is the reduction of copper ion, Cu to Cu, by protein amides under alkaline conditions. In the Lowry method the reduced copper— and to a lesser extent some protein side chains— react with the Folin-Ciocalteu reagent (phosphomolybdate/pho-sphotungstate) to produce a blue color. In the BCA assay the BCA complexes with the Cu, which absorbs strongly at 562 nm. Although both the Lowry and BCA methods must be carefully timed and are subject to interference from buffer components, the BCA method is less susceptible to interference, especially by detergents. 

We have prepared an ATF solution using a protocol similar to those previously reported (7, 15). Our preparation contained three of the major proteins found in tears and lens deposits, namely, lysozyme, albumin and mucin. Hydrogels prepared from HEMA, monomers 1 or 2, or their mixed combinations were incubated in ATF for 24h at 36 °C, and the adsorbed proteins were quantified using the BCA assay (7). The results of the study are presented in Figure 2. 

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