BCA法测蛋白含量

THE BICINCHONINIC ACID (BCA) ASSAY FOR DETERMINATION OF TOTAL PROTEINPrinciple原理Smith et al. (1985) introduced the bicinchoninic acid (BCA) protein assay reagent. In one sense, it is a modification of the Lowry protein assay reagent. The mechanism of color formation with protein for the BCA protein assay reagent is similar to that of the Lowry reagent, but there are several significant differences. The BCA protein assay reagent combines the reduction of Cu2+ to Cu+ by protein in an alkaline medium (i.e., the biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+) by bicinchoninic acid. The purple-colored reaction product of this method is formed by the chelation of two molecules of BCA with one cuprous ion (Fig. A.3H.3). The BCA/copper complex is water-soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations. The primary advantage of the BCA protein assay reagent is that most surfactants, even if present in the sample at concentrations up to 5% (v/v), are compatible with this method. Table A.3H.2 is a brief troubleshooting guide for this technique.二喹啉甲酸（BCA）法是在福林酚法的基础上改善而来的，即在碱性条件下蛋白质将二价铜离子还原成一价铜离子，然后与BCA试剂反应生成紫色化合物，在562 nm处检测。Materials试剂材料Protein standard: 2 mg/mL BSA20 mg BSA in 10 mL of 0.9% NaCl containing 0.05% (w/v) sodium azide. Store up to 6 months at 4 C.20 mg牛血清蛋白 + 10 mL含有0.9% NaCl和0.05% (w/v)的叠氮钠。4 C放置6个月。Sample buffer or solventProtein sampleBCA reagent A:1 g 4,4 -dicarboxy-2,2 -biquinoline, disodium salt (Na2BCA; 1% w/v final)二喹啉甲酸二钠盐2 g Na2CO3H2O (2% w/v final)一水合碳酸钠160 mg sodium tartrate dihydrate (0.16% w/v final)二水合酒石酸钠0.4 g NaOH (0.4% w/v final)氢氧化钠0.95 g NaHCO3 (0.95% w/v final)碳酸氢钠Dissolve all of the above chemicals except the sodium bicarbonate in deionized water and adjust the final volume to 100 mL. Adjust the pH to 11.25 by adding the sodium bicarbonate a little at a time. Store this alkaline reagent in a plastic container 1 to 3 weeks at room temperature, longer at 4 C.除了碳酸氢钠，把其他试剂都加入100 mL去离子水中，然后分次加碳酸氢钠，一次加一点，调pH到11.25。试剂储存于塑料瓶中常温1-3周，4 C可放置更久。Only the disodium salt of BCA is soluble at neutral pH; the free acid is not readily soluble.只有二钠盐BCA在中性条件下可溶；其他形式溶解性不好。BCA reagent B:4 g CuSO45H2O (4% w/v final) in 100 mL H2O. Store up to 6 months at room temperature.4 g 五水硫酸铜 + 100 mL 去离子水。室温可放置6个月。BCA working reagent: Mix 100 parts BCA reagent A with 2 parts reagent B.将100份的BCA试剂A和2份的BCA试剂B混合起来。Procedures过程1. Prepare a dilution series of 2 mg/mL BSA in sample buffer or diluent to cover a range from 125 to 2000 g/mL.稀释2 mg/mL BSA制备浓度范围1252000 g/mL。2. Add 100 L sample, diluted standard, or buffer (blank) into appropriately labeled tubes.在试管中加100 L的样品、标准溶液和空白。3. Add 2 mL BCA working reagent mix to each tube. Vortex immediately.加2 mL BCA工作液至每个试管，迅速混匀。4. Incubate samples and standards for 30 min at 37 C, then cool to room temperature.将样品和标准溶液及空白在37 C保温30分钟，然后冷却至室温。5. Measure the color at 562 nm (A 562) on a spectrophotometer zeroed with deionized water.以空白调零，在562 nm处测吸光值。6. Plot a standard curve by graphing the average net or blank-corrected A562 values for the standards versus protein concentration in micrograms per milliliter. Example color response curves for BSA and BGG are shown in Fig. A.3H.4.以校正过的吸光值A5