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    普利莱
  • 货  号:

    P1511-3
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  • 引用文献

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+BCACu1+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。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微板比色仪,最佳工作波长562nm,可540-590nm之间。

工作溶液(Working Reagent, WR)配制:50体积BCA Reagent1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25 µl连续倍比稀释得到BSA标准溶液16008004002001005025 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8001000 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,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,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 (540-590 nm之间)光密度(OD)

4. 绘制标准曲线X轴为BSA标准蛋白浓度(mg/mlµg/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。


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 30min25ºC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10min内可以测定30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检测样品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 #P1512EDTA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、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


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%

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