THE BICINCHONINIC ACID (BCA) ASSAY FOR DETERMINATION OFTOTAL PROTEIN
Principle原理
Smith et al. (1985) introduced the bicinchoninic acid (BCA) protein assay reagent. Inone sense, it is a modification of the Lowry protein assay reagent. The mechanism ofcolor formation with protein for the BCA protein assay reagent is similar to that ofthe Lowry reagent, but there are several significant differences. The BCA protein assayreagent combines the reduction of Cu2+ to Cu+三星广告背景音乐 by protein in an alkaline medium (i.e., thebiuret reaction) with the highly sensitive and selective colorimetricdetection of the cuprous cation (Cu+) by bicinchoninic acid.The purple-colored reactionproduct of this method is formed by the chelation of two molecules of BCA with onecuprous ion (Fig. A.3H.3). The BCA/copper complex is water-soluble and exhibits astrong linear absorbance at 562 nm with increasing protein concentrations. The primaryadvantageoftheBCAproteinassayreagentisthatmostsurfactants,evenifpresentinthesample
at concentrations up to 5% (v/v), are compatible with this method. Table A.3H.2is 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试剂反应生成紫化合物，在562 nm处检测。
Materials试剂材料
Protein standard:2 mg/mL BSA
20 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。
流星雨 歌词Sample buffer or solvent
Protein sample
BCA 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 Na2CO3·H2O (2% w/v final)一水合碳酸钠
刘流的老婆
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 deionizedwater and
adjust the final volume to 100 mL. Adjust the pH to 11.25 by addingthe sodium bicarbonate a little at a time. Store this alkaline reagent in a plasticcontainer 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 CuSO4·5H2O (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 rangefrom 125 to 2000 μg/mL.
稀释2 mg/mL BSA制备浓度范围125~2000 μg/mL。
2. Add 100 μL sample, diluted standard, or buffer (blank) into appropriately labeledtubes.
在试管中加100 μL的样品、标准溶液和空白。港囧背景音乐
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.
适合跳爵士舞的音乐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 deionizedwater.
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 forthe 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A562做曲线，蛋白浓度单位为μg/mL。以BSA或BGG做标准曲线的例子见图A.3H.4。
7. Determine the protein concentration of the sample by interpolation from the plot(see Strategic Planning).
后弦新歌根据标准曲线的公式进行蛋白质浓度的计算。