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[Bio101] BCA (Bicinchoninic Acid) Protein Assay
[Bio101] BCA(二喹啉甲酸)蛋白定量法   

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Abstract

The BCA protein assay is used for quantitation of total protein in a sample. The principle of this method is that proteins can reduce Cu+2 to Cu+1 in an alkaline solution (the biuret reaction) and result in a purple color formation by bicinchoninic acid. The reduction of copper is mainly caused by four amino acid residues including cysteine or cystine, tyrosine, and tryptophan that are present in protein molecules. However, unlike the Coomassie dye-binding methods, the universal peptide backbone also contributes to color formation, helping to minimize variability caused by protein compositional differences. Compared to the Bradford assay, the BCA assay is more objective since the universal peptide backbone also contributes to color formation. One disadvantage of the BCA assay compared to the Bradford assay is that it is susceptible to interference by some chemicals present in protein samples, including reducing agents (i.e., DTT and beta—mercaptoethanol), copper chelators (i.e., EDTA, EGTA) and buffers with high concentration, which can be avoided by generating diluted samples.

Keywords: Protein concentration(蛋白浓度), Protein measument(蛋白质的测定), BSA standards(BSA标准), Bradford assay(Bradford法), Reduction of copper(还原铜)

Materials and Reagents

  1. Bovine serum abumin (BSA) (Sigma)
  2. BCA protein assay reagents (Pierce, catalog number: 23227 )
  3. BCA working reagent (WR)

Equipment

  1. Spectrophotometer (Tecan)

Procedure

  1. Prepare bovine serum abumin (BSA) standards. Prepare 1 ml of BSA stock (2 mg ml-1, dissolved in H2O) and then make serial (5-8) dilutions with a range of 20-2,000 μg ml-1.

  2. Prepare BCA working reagent (WR). Calculate the total volume of WR needed. Prepare WR by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50: 1, Reagent A: B) (the mixture appears to be clear and green solution).

  3. For test-tube measurement, 2.0 ml of the WR is required for each sample. Sample to WR ratio is 1: 20.
    1. Pipette 0.1 ml of each standard and unknown protein sample replicate into an appropriately labeled test tube. Set two blank tubes in duplicate. For a standard curve, add 0.1 ml H2O instead of BSA solution. For the protein samples, add 0.1 ml protein preparation buffer.
    2. Add 2.0 ml of the WR to each tube and mix well.
    3. Cover and incubate tubes at 37 °C for 30 min.
    4. Keep all tubes at RT for 10 min before measurement.
    5. Take absorbance readings at 562 nm.

  4. For microplate measurement, 200 μl of WR reagent is required. Sample to WR ratio is 1: 8 or 1: 20 (when sample is limited).
    1. Pipette 25 or 10 μl of each standard or protein sample replicate into a microplate well. Use water or protein sample preparation buffer as blank solutions for standard curve and protein samples, respectively.
    2. Add 200 μl of the WR to each well.
    3. Cover and incubate tubes at 37 °C for 30 min.
    4. Keep all tubes at RT for 10 min before measurement.
    5. Take absorbance readings at 562 nm on a plate reader.

Acknowledgments

This work was done in the Andrew Binns Lab in the Department of Biology at University of Pennsylvania, USA and supported by National Science Foundation grants MCB 0421885 and IOS-0818613.

References

  1. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. and Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Anal Biochem 150(1): 76-85.
  2. Instructions for BCA Protein Assay Kit from Thermo Scientific.

简介

BCA蛋白测定用于定量样品中的总蛋白。该方法的原理是蛋白质可以在碱性溶液(缩二脲反应)中将Cu 2+还原为Cu 2+ +1并且导致通过二喹啉酸形成紫色。铜的还原主要由存在于蛋白质分子中的四个氨基酸残基(包括半胱氨酸或胱氨酸,酪氨酸和色氨酸)引起。然而,与考马斯染料结合方法不同,通用肽骨架也有助于颜色形成,有助于使由蛋白质组成差异引起的可变性最小化。与Bradford测定相比,BCA测定更客观,因为通用肽骨架也有助于颜色形成。与Bradford测定相比,BCA测定的一个缺点是其易受蛋白质样品中存在的一些化学物质的干扰,包括还原剂(例如DTT和β-巯基乙醇),铜螯合剂>即EDTA,EGTA)和具有高浓度的缓冲液,通过产生稀释的样品可以避免。

关键字:蛋白浓度, 蛋白质的测定, BSA标准, Bradford法, 还原铜

材料和试剂

  1. 牛血清白蛋白(BSA)(Sigma)
  2. BCA蛋白测定试剂(Pierce,目录号:23227)
  3. BCA工作试剂(WR)

设备

  1. 分光光度计(Tecan)

程序

  1. 制备牛血清白蛋白(BSA)标准品。 制备1ml的BSA储备液(2mg ml溶于H 2 O中),然后进行系列(5-8)稀释,范围为20-2,000 μgml -1

  2. 准备BCA工作试剂(WR)。 计算所需的WR的总体积。 通过混合50份BCA试剂A与1份BCA试剂B(50:1,试剂A:B)(混合物看起来是澄清的绿色溶液)来制备WR。
  3. 对于试管测量,每个样品需要2.0ml的WR。 样品与WR的比率为1:20。
    1. 移取0.1ml的每种标准和未知的蛋白质样品复制到适当标记的试管中。 设置两个空白管一式两份。 对于标准曲线,加入0.1ml H 2 O而不是BSA溶液。 对于蛋白质样品,加入0.1ml蛋白质制备缓冲液。
    2. 向每个管中加入2.0ml的WR,并混匀
    3. 盖并孵育管在37℃下30分钟
    4. 测量前,将所有试管在室温下保持10分钟
    5. 吸光度读数为562 nm。

  4. 对于微孔板测量,需要200μl的WR试剂。 样品与WR比为1:8或1:20(当样品有限时)。
    1. 移液器25或10μl的每个标准品或蛋白质样品复制到微孔板孔中。 使用水或蛋白质样品制备缓冲液分别作为标准曲线和蛋白质样品的空白溶液
    2. 向每个孔中加入200μl的WR
    3. 盖并孵育管在37℃下30分钟
    4. 测量前,将所有试管在室温下保持10分钟
    5. 在读板器上读取562 nm处的吸光度读数

参考文献

  1. Smith,P.K.,Krohn,R.I.,Hermanson,G.T.,Mallia,A.K.,Gartner,F.H.,Provenzano,M.D.,Fujimoto,E.K.,Goeke,N.M.,Olson,B.J.and Klenk,D.C。(1985)。 使用bicinchoninic acid测量蛋白质。 Anal Biochem 150 (1):76-85
  2. Thermo Scientific的BCA蛋白检测试剂盒说明
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:He, F. (2011). BCA (Bicinchoninic Acid) Protein Assay. Bio-protocol Bio101: e44. DOI: 10.21769/BioProtoc.44;
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Akhtar Ali
School of Medicine GNU South Korea
How Can we use The tecan infinite m200 . Any User Manual
4/9/2015 6:48:53 PM Reply
daniel wasswa
london metropolitan university

hi fanglian , i am under taking a research project which involves the evaluation of protein membrane vesicles in erythrocytes , would the same principle apply to my blood sample as in i have to make a several dilution factors ??

11/16/2015 6:20:57 AM


María Teresa Fernández
Unach
necesito determinar cuanto de proteína tengo en mi muestra;
cuanta muestra necesito para poder utilizar este método o como se que tanto de mi muestra utilizar en este método?
1/12/2015 10:39:25 PM Reply
Fanglian He
University of Pennsylvania

Hi Maria,

Would you lease put your question in English? Thanks.

1/20/2015 1:42:56 PM


María Teresa Fernández
Unach

I need to determine how much protein I have in my sample;
How much sample need to use this method or as both of my sample used in this method?

I'm not good in english sorry, but is important for me know that both of my sample can process this method if I have only 1mg can used this method.

I explain?

1/24/2015 5:55:54 AM


Fanglian He
University of Pennsylvania

Hi, Maria,

My apologies for the late response. To determine how much sample is used for BCA protein assay, you should first make several dilutions of your sample, like 1:10, 1:20, 1:50... (if your sample is limited, you can start with a higher dilution. The absorbance readings (at 562 nm) which fall into the linear section of the standard curve can be used to calculate the concentration of your undiluted protein sample.

Hope my answer helps. Please let me know if anything is unclear to you.

Good luck,
Fanglian

3/7/2015 12:18:17 AM