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[Bio101] Bradford Protein Assay
[Bio101] Bradford蛋白定量法   

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Abstract

The Bradford protein assay is used to measure the concentration of total protein in a sample. The principle of this assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue. This method actually measures the presence of the basic amino acid residues, arginine, lysine and histidine, which contributes to formation of the protein-dye complex. Unlike the BCA assay, reducing agents (i.e., DTT and beta—mercaptoethanol) and metal chelators (i.e., EDTA, EGTA) at low concentration do not cause interference. However, the presence of SDS even at low concentrations can interfere with protein-dye binding. This technique was invented by Bradford (1976).

Materials and Reagents

  1. Bovine Serum Abumin (BSA) (Sigma-Aldrich)
  2. Coomassie Brilliant Blue G-250 (Sigma-Aldrich, catalog number: 27815 )
  3. Methanol
  4. Phosphoric acid (H3PO4)
  5. Bradford reagent (see Recipes)

Equipment

  1. Spectrophotometer (Tecan)
  2. Whatman #1 paper (Whatman)

Procedure

  1. Standard assay procedure (for sample with 5-100 µg ml-1 protein)
    1. Prepare five to eight dilutions of a protein (usually BSA) standard with a range of 5 to 100 µg protein.
    2. Dilute unknown protein samples to obtain 5-100 µg protein/30 µl.
    3. Add 30 µl each of standard solution or unknown protein sample to an appropriately labeled test tube.
    4. Set two blank tubes. For the standard curve, add 30 µl H2O instead of the standard solution. For the unknown protein samples, add 30 µl protein preparation buffer instead. Protein solutions are normally assayed in duplicate or triplicate.
    5. Add 1.5 ml of Bradford reagent to each tube and mix well.
    6. Incubate at room temperature (RT) for at least 5 min. Absorbance will increase over time; samples should incubate at RT for no more than 1 h.
    7. Measure absorbance at 595 nm.

  2. Microassay procedure (<50 µg ml-1 protein):
    1. Prepare five standard solutions (1 ml each) containing 0, 10, 20, 30, 40 and 50 µg ml-1 BSA
    2. Pipet 800 μl of each standard and sample solution (containing for <50 µg ml-1 protein) into a clean, dry test tube. Protein solutions are normally assayed in duplicate or triplicate.
    3. Add 200 μl of dye reagent concentrate to each tube and vortex.
    4. Follow the procedure described above for the standard assay procedure.

Recipes

  1. Bradford reagent
    Dissolve 50 mg of Coomassie Brilliant Blue G-250 in 50 ml of methanol and add 100 ml 85% (w/v) phosphoric acid (H3PO4).
    Add the acid solution mixture slowly into 850 ml of H2O and let the dye dissolve completely (note: Do not add H2O into the acid solution).
    Filter using Whatman #1 paper to remove the precipitates just before use.
    Store in a dark bottle at 4 °C.

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. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  2. Stoscheck, C. M. (1990). Quantitation of protein. Methods Enzymol 182: 50-68.

简介

Bradford蛋白测定用于测量样品中总蛋白的浓度。 该测定的原理是在酸性条件下蛋白质分子与考马斯染料的结合导致从棕色到蓝色的颜色变化。 该方法实际上测量了有助于形成蛋白质 - 染料复合物的碱性氨基酸残基,精氨酸,赖氨酸和组氨酸的存在。 与BCA测定不同,低浓度的还原剂(即DTT和β-巯基乙醇)和金属螯合剂(即EDTA,EGTA)不引起干扰。 然而,甚至在低浓度的SDS的存在可以干扰蛋白质 - 染料结合。

材料和试剂

  1. 牛血清白蛋白(BSA)(Sigma-Aldrich)
  2. 考马斯亮蓝G-250(Sigma-Aldrich,目录号:27815)
  3. 甲醇
  4. 磷酸(H 3 PO 4)
  5. Bradford试剂(见配方)

设备

  1. 分光光度计(Tecan)
  2. Whatman#1论文(Whatman)

程序

  1. 标准测定程序(对于具有5-100μgml -1 蛋白质的样品)
    1. 准备5-8个稀释度的蛋白质(通常为BSA)标准品,范围为5-100μg蛋白质
    2. 稀释未知蛋白样品,获得5-100μg蛋白/30μl。
    3. 加入30μl每个标准溶液或未知蛋白样品到适当标记的试管。
    4. 设置两个空白管。 对于标准曲线,加入30μlH 2 O 2而不是标准溶液。 对于未知的蛋白质样品,加入30μl蛋白制备缓冲液。 蛋白质溶液通常一式两份或三份进行测定
    5. 向每个管中加入1.5ml Bradford试剂并混匀。
    6. 在室温(RT)孵育至少5分钟。 吸光度将随时间增加; 样品应在室温下孵育不超过1小时
    7. 测量595nm处的吸光度

  2. 微量分析程序(<50μgml -1 sup蛋白):
    1. 制备含有0,10,20,30,40和50μg/ml BSA的五种标准溶液(每种1ml)
    2. 吸取800μl的每种标准品和样品溶液(含有<50μg/ml的sup-1蛋白质)到干净,干燥的试管中。 蛋白质溶液通常一式两份或三份进行测定
    3. 向每个管中加入200μl染料试剂浓缩液并涡旋
    4. 按照上述标准测定程序的步骤

食谱

  1. Bradford试剂
    将50mg考马斯亮蓝G-250溶于50ml甲醇中并加入100ml 85%(w/v)磷酸(H 3 PO 4)。
    将酸溶液混合物缓慢加入到850ml H 2 O中,使染料完全溶解(注意:不要加入H 2 O into the acid solution )。
    在使用前,使用Whatman#1滤纸过滤以除去沉淀。
    储存在4°C的深色瓶中。

参考文献

  1. Bradford,M.M。(1976)。 利用蛋白质染料结合原理的快速灵敏的微克量蛋白定量方法 。 Anal Biochem 72:248-254。
  2. Stoscheck,C.M。(1990)。 蛋白质定量。方法Enzymol 182:50- 68.
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:He, F. (2011). Bradford Protein Assay. Bio-protocol Bio101: e45. DOI: 10.21769/BioProtoc.45;
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jasim al-sede
university of Bucharest
How to prepare BSA 1mM by using 0.660 g
11/22/2017 9:47:43 AM Reply
jasim al-sede
university of Bucharest
Hello, I am a new subscriber jasim
11/22/2017 9:43:14 AM Reply
Sneha More
Ph.D. student
hi every one , i am facing problem is plotting std protein graph. here i used 1mg/ml std protein. different concentration used for plotting is 0.2mg / 4ml 0. 4mg/ 4ml 0.6mg/ 4ml 0.8 mg/ 4ml and 1mg /4ml. and for estimation of protein only 50 ul sample was used form each above concentration for protein estimation than 2.5 ml of Bradford reagent was added to each tube and read at 595.. what concentration of protein is should take on X axis to plot a std protein graph. is it 0.2mg / 4ml 0. 4mg/ 4ml 0.6mg/ 4ml 0.8 mg/ 4ml 1mg /4ml. or according to 50 ul what ever protein concentration will be, that i need to used on X axis ? please suggest. i need to plot a std graph of protein but i followed above protocol, please help
10/20/2017 2:47:35 AM Reply
mahmoud ali
agriculture research center egypt
Bradford reagent
could i use it after preparation or must incubate over night before using
the volume of Bradford reagent 1.5 ml and 30 µl for sample ???
2/14/2017 9:25:12 AM Reply
mahmoud ali
agriculture research center egypt
can i replace phosphoric with orthophosphoric acid?
and if i can could i use it without dilution direct from bottle
2/11/2017 1:55:15 AM Reply
Fanglian He
University of Pennsylvania

Hi,

Phosphoric acid and orthophosphoric acid are often considered as the same thing (http://www.sigmaaldrich.com/catalog/product/aldrich/345245?lang=en®ion=US).

The one mentioned in the protocol was 85% (w/v) from vendor. I did not dilute it. Whatever the concentration of your H3PO4 stock is, its final concentration in the reagent should be 8.5% (w/v).

Hope this helps.
Fanglian


2/14/2017 12:51:57 AM


Le Nghia
Laboratory of Molecular Biotechnology
In the dye reagent's recipe, I wonder how much is the total volume of reagent. Is it about 150 ml, include 50 mg Coomassie G250, 50 ml Methanol, and 100 ml 85% Phosphoric acid? Don't we need to add mQ to adjust final volume?
And I think Methanol is poisonous, so can I replace it by Ethanol?
Thanks.
10/19/2016 6:41:36 PM Reply
Fanglian He
University of Pennsylvania

Hi Le,

Sorry to miss your questions. Yes, you were right that we should adjust the final vol.. As 50mg Coomassie G250 would not change the vol significantly, I did not write it in that way.

That is good point. I never got chance to try Ethanol. Please share your experience here if it works in your hand.

Thanks,
Fanglian

2/14/2017 12:57:35 AM


Le Nghia
Laboratory of Molecular Biotechnology
In the dye reagent's recipe, I wonder how much is the total volume of reagent. Is it about 150 ml, include 50 mg Coomassie G250, 50 ml Methanol, and 100 ml 85% Phosphoric acid? Don't we need to add mQ to adjust final volume?
And I think Methanol is poisonous, so can I replace it by Ethanol?
Thanks.
10/19/2016 6:41:35 PM Reply
nebula PH
gorgan university of medical science
hi dears
im using SDS in my lysis buffer so it could make problem for my bradford assay?
4/15/2015 12:19:07 PM Reply
Fanglian He
Department of Biology, University of Pennsylvania, USA

Hi,

Yes, it could cause the problem. You may try BCA protein assay instead.

Good luck,
Fanglian

4/17/2015 12:03:06 AM


Wynifred Wang
China Aguriculral University(CAU,China)
Vary Good.
12/5/2013 7:11:38 AM Reply
Alaa Elminisy
National research center

Hi dears,
Bradford reagent used diluted(1x) or 5x??

12/9/2015 12:21:48 AM


Fanglian He
University of Pennsylvania

Hi Alaa,

It is 1x.

2/14/2017 1:02:08 AM