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描述：Bicinchoninic acid (BCA )法是近来广为应用的蛋白定量方法。其原理与Lowery法蛋白定量相似，即在碱性环境下蛋白质与Cu2+络合并将Cu2+还原成Cu1+。BCA与Cu1+结合形成稳定的紫蓝色复合物，在562 nm处有高的光吸收值并与蛋白质浓度成正比，据此可测定蛋白质浓度。与Lowery法相比，BCA蛋白测定方法灵敏度高，操作简单，试剂及其形成的紫蓝色复合物稳定性俱佳，并且受干扰物质影响小。与Bradford法相比，BCA法的显著优点是不受去垢剂的影响。
组成与储存：(500次)
(1) BCA Reagent 100ml，室温保存；
(2) Cu Reagent 2.5ml，室温保存；
(3) BSA standard 4mg/ml 1ml，−20ºC冻存。12个月有效。
所需设备：比色计、酶标仪或微板比色仪，最佳工作波长562nm，可在540-590nm之间。
工作溶液(Working Reagent, WR)配制：将50体积BCA Reagent与1体积Cu Reagent混合即为WR工作试剂，呈嫩绿色；室温1周内稳定。
标准蛋白溶液配制：用双蒸水、0.9%生理盐水、PBS或与待测蛋白样品匹配的缓冲液进行倍比稀释: 20µl 4000µg/ml BSA + 30 µl稀释溶液(H2O/PBS/0.9%NaCl) = 50µl (BSA=1600 µg/ml)，从中取25 µl连续倍比稀释，得到BSA标准溶液1600、800、400、200、100、50、25 µg/ml，各25 µl。通常，样品蛋白浓度不会太高，也可以预先稀释待测样品，可以省略1600 µg/ml标准管而直接从800或1000 µg/ml开始，能节省标准蛋白用量。
蛋白浓度测定：
蛋白质浓度线性检测范围为10-2000 µg/ml。标准测定时，用1 cm光程玻璃或塑料比色皿，反应终体积1.1 ml，比色计测定。微板测定时，用96孔板，反应终体积225 µl，用酶标仪、微板比色仪测定。
1. 标准测定：将0.05~0.1 ml标准品或待测样本与1 ml WR工作溶液混合。微板测定：将25 µl标准品或待测样本与200 µl WR工作溶液混合。
2. 37ºC反应30min；也可25ºC室温2小时或过夜。60ºC 30 min反应可增加检测灵敏度至5-250µg/ml。
3. 将反应管冷却至室温。测定562 nm (可在540-590 nm之间)光密度(OD)值。
4. 绘制标准曲线。X轴为BSA标准蛋白浓度(mg/ml或µg/ml)，Y轴为各标准管对应的OD562值。用Excel拟合曲线并计算蛋白浓度。
表1 标准测定和微板测定方案的加样量和比例
|
|
微板(microplate)测定方案 |
标准比色杯测定方案 |
|||
|
标号 |
蛋白浓度(µg/ml) |
标准或待测蛋白体积 (µl) |
WR工作试剂(µl) |
标准或待测蛋白体积 (ml) |
WR工作试剂(ml) |
|
1 |
0 |
25 |
200 |
0.05-0.1 |
1 |
|
2 |
25 |
25 |
200 |
0.05-0.1 |
1 |
|
3 |
50 |
25 |
200 |
0.05-0.1 |
1 |
|
4 |
100 |
25 |
200 |
0.05-0.1 |
1 |
|
5 |
200 |
25 |
200 |
0.05-0.1 |
1 |
|
6 |
400 |
25 |
200 |
0.05-0.1 |
1 |
|
7 |
800 |
25 |
200 |
0.05-0.1 |
1 |
|
8 |
1600 |
25 |
200 |
0.05-0.1 |
1 |
|
待测样品 |
25 |
200 |
0.05-0.1 |
1 |
|
注意事项
1. 37ºC 30min或25ºC室温反应2小时对测量较为便利，但严格来讲此时反应尚未达到终点，通常每10 min OD562值升高约2.3%。然而，通常在10min内可以测定30管并不会明显影响测定精度。
2. BCA法检测范围为20-2000 µg/ml。检测0.5~10 µg/ml高度稀释蛋白样品应采用Bradford法蛋白质定量试剂盒(# P1510)。60ºC 30 min反应可增加检测灵敏度至5~250µg/ml。
3. BCA法检测样品中含有脂类物质时光吸收值会偏高。样品中EDTA或葡萄糖浓度大于10 mM不能使用BCA方法，葡萄糖浓度大于10 mM时可用改良Lowry法蛋白定量试剂盒 #P1512，EDTA大于10 mM的样品可用Bradford法蛋白质定量试剂盒(# P1510)。另外，蛋白质样品经液体样品蛋白抽提试剂(#P1255)沉淀后，可彻底去除干扰BCA法、Bradford法、和Lowry法蛋白测定的物质。
4. 欲使测量能耐受下面表2所提示的最大干扰物质浓度，并保持测量精度，应在蛋白标准管中加入相应浓度的干扰物质，但会给操作带来不便。
5. 可测量吸附于固相支持物乳酶标板、琼脂糖、亲和层系凝胶上的蛋白。
6. 每次测定应该重新测定并制作标准曲线。
参考文献：
Smith P et al, 1995, Measurment of protein using bicinchiconic acid, Anal. Biochem. 150, 76-85
表2 BCA法物质干扰及耐受的最大浓度
|
Buffer Systems |
Sodium phosphate 25 mM |
|
Bicine, pH 8.4 20 mM |
Sucrose 40% |
|
Bis-Tris, pH 6.5 33 mM |
Sodium ortho-Vanadate in PBS, pH 7.2, 1 mM |
|
Calcium chloride in TBS, pH 7.2 10 mM |
Urea 3 M |
|
CHES, pH 9.0 100 mM |
Chelating agents |
|
Cobalt chloride in TBS, pH 7.2 0.8 M |
EDTA 10 mM |
|
Ferric chloride in TBS, pH 7.2 10 mM |
EGTA,any level, not compatible |
|
HEPES 100 mM |
Sodium citrate 200 mM |
|
MOPS, pH 7.2 100 mM |
Detergents |
|
Nickel chloride in TBS 10 mM |
Brij-35 5% |
|
PBS; no interference |
Brij-52 1% |
|
NaCl (0.15 M), pH 7.2, no interference |
CHAPS 5% |
|
PIPES, pH 6.8 100 mM |
CHAPSO 5% |
|
Sodium acetate, pH 4.8 200 mM |
Deoxycholic acid 5% |
|
Sodium citrate, pH 4.8 or pH 6.4 200 mM |
Nonidet P-40 (Igepal CA-630) 5% |
|
Tricine, pH 8.0 25 mM |
SDS 5% |
|
Triethanolamine, pH 7.8 25 mM |
Span 20 1% |
|
Tris 250 mM |
Triton X-100 5% |
|
TBS buffer, no interference |
Triton X-114 1% |
|
1 x SDS-PAGE loading buffer, no interference |
Tween-20 5% |
|
Zinc chloride (10 mM) in TBS, pH 7.2, 10 mM |
Tween-60 5% |
|
Buffer Additives |
Tween-80 5% |
|
Ammonium sulfate 1.5 mM |
Zwittergents 1% |
|
Aprotinin 10 mg/L |
Reducing & Thiol Containing Agents |
|
Glucose 10 mM |
Dithioerythritol (DTE) 1 mM |
|
Glycerol 10% |
Dithiothreitol (DTT) 1 mM |
|
Guanidine•HCl 4 M |
2-Mercaptoethanol 1 mM |
|
HCl 100 mM |
Tributyl Phosphine 0.01% |
|
Imidazole 50 mM |
Solvents |
|
Leupeptin 10 mg/L |
Acetone 10% |
|
PMSF 1 mM |
Acetonitrile 10% |
|
Sodium azide 0.20% |
DMF 10% |
|
Sodium bicarbonate 100 mM |
DMSO 10% |
|
Sodium chloride 1 M |
Ethanol 10% |
|
Sodium hydroxide 100 mM |
Methanol 10% |
目前，使用普利莱BCA蛋白定量试剂盒发表的SCI文章已超过百篇，部分文章如下，供参考。
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