BCA蛋白(bái)濃度測定試劑盒（目錄号：RTP7102）
BCA Protein Assay Kit
● 試劑盒内容及保存：
|
貨号
|
産品名稱
|
包裝
|
貯存
|
|
RTP7102-01
|
BCA試劑A
|
100 ml
|
RT
|
|
RTP7102-02
|
BCA試劑B
|
3 ml
|
RT
|
|
BSA-01
|
牛血清白(bái)蛋白(bái)(BSA)标準溶液（5 mg/ml）
|
2×1 ml
|
-20℃
|
|
RT0280-02
|
PBS溶液
|
10 ml
|
4℃
|
|
|
說(shuō)明書(shū)
|
1份
|
|
● 儲存條件(jiàn)和效期：
試劑A和試劑B室溫貯存；牛血清白(bái)蛋白(bái)标準溶液-20℃貯存；PBS溶液4℃貯存。本試劑盒有效期1年(nián)。
● 産品簡介：
BCA蛋白(bái)濃度測定試劑盒的原理(lǐ)是蛋白(bái)質分(fēn)子中肽鍵結構在堿性環境下能與Cu2+生(shēng)産絡合物，并将Cu2+還(hái)原成Cu+，而BCA試劑可(kě)以特異性地與Cu+結合，形成穩定的有顔色的複合物，并在562nm處有最大(dà)的光(guāng)吸收值，該複合物顔色的深淺與蛋白(bái)質濃度成正比，可(kě)以根據吸收值的大(dà)小來(lái)測定蛋白(bái)的含量。
本試劑盒可(kě)以檢測500個樣品（使用微孔闆）或50個樣品（使用試管）。
● 産品特點：
1. 靈敏度高，檢測濃度下限達到25μg/ml（在20-1000μg/ml濃度範圍内有較好的線性關系），最小檢測蛋白(bái)量達到0.2μg，待測樣品體(tǐ)積爲1-20μl。
2. BCA法測定蛋白(bái)濃度的最大(dà)優點是蛋白(bái)濃度的測定可(kě)以耐受高濃度的去(qù)垢劑，可(kě)以兼容樣品中高達5%的SDS，5%的Triton
X-100，5%的Tween
20, 60, 80。但(dàn)受螯合劑和略高濃度的還(hái)原劑的影(yǐng)響，需确保EDTA低于10mM，無EGTA，二硫蘇糖醇低于1mM，β-巯基乙醇低于0.01%。
● 操作(zuò)方法
BCA工(gōng)作(zuò)液配制：
将試劑A和試劑B按照(zhào)體(tǐ)積比50:1比例混合，配成BCA工(gōng)作(zuò)液。
如(rú)，取50ml 試劑A與1ml試劑B混合，配成51 ml BCA工(gōng)作(zuò)液。兩者混合時會有沉澱形成，徹底混勻後沉澱消失，溶液應爲澄清淡藍色溶液。
注：BCA工(gōng)作(zuò)液室溫可(kě)放(fàng)置一周不失效。
微孔闆測定程序：（工(gōng)作(zuò)範圍20-2000 μg/ml）
1. 蛋白(bái)标準品配制：室溫完全溶解蛋白(bái)标準品，取20μl
5mg/ml BSA蛋白(bái)标準溶液用PBS溶液稀釋至100μl，使其終濃度爲1.0 mg/ml。
2.
按照(zhào)下表配制BSA标準測定溶液：
|
編号
|
0
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
|
|
1 mg/ml BSA 标準溶液 μl
|
5 mg/ml BSA 标準溶液 μl
|
|
BSA标準溶液 μl
|
0
|
0.5
|
2.5
|
5.0
|
10
|
15
|
20
|
6
|
8
|
|
PBS 溶液 μl
|
20
|
19.5
|
17.5
|
15
|
10
|
5
|
0
|
14
|
12
|
|
BSA終濃度 μg/ml
|
0
|
25
|
125
|
250
|
500
|
750
|
1000
|
1500
|
2000
|
|
總體(tǐ)積 μl
|
20 μl
|
3.
将适當體(tǐ)積的待測樣品加入到微孔闆中，并用PBS補足到20
μl
4.
向微孔闆中加入200
μl BCA工(gōng)作(zuò)液，混勻，37℃放(fàng)置30分(fēn)鍾；
注：也可(kě)以室溫放(fàng)置2小時，或60℃放(fàng)置30分(fēn)鍾。BCA法測定蛋白(bái)濃度時，顔色會随着時間的延長不斷加深。并且顯色反應會因溫度升高而加快(kuài)。如(rú)果濃度較低，适合在較高溫度孵育，或适當延長孵育時間。
5.
測定562
nm 處的吸光(guāng)值，并記錄讀(dú)數；以不含BSA
的樣品的光(guāng)吸收值作(zuò)爲空白(bái)對照(zhào)。
6. 以A562爲縱坐(zuò)标，BSA含量爲橫坐(zuò)标，繪制标準曲線，計(jì)算樣品中的蛋白(bái)濃度。如(rú)果所得(de)到的蛋白(bái)濃度不在标準曲線範圍内，請(qǐng)稀釋樣品後重新測定。
試管測定程序：（工(gōng)作(zuò)範圍20-1000
μg/ml）
1. 蛋白(bái)标準品配制：室溫完全溶解蛋白(bái)标準品，取150μl
5mg/ml BSA蛋白(bái)标準溶液，加入600μl PBS溶液稀釋至750μl，使其終濃度爲1.0 mg/ml。
2.
按照(zhào)下表配制BSA标準測定溶液：
|
編号
|
0
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
|
|
1 mg/ml BSA 标準溶液 μl
|
5 mg/ml BSA 标準溶液 μl
|
|
BSA标準溶液 μl
|
0
|
2.5
|
12.5
|
25
|
50
|
75
|
100
|
30
|
40
|
|
PBS 溶液 μl
|
100
|
97.5
|
87.5
|
75
|
50
|
25
|
0
|
70
|
60
|
|
BSA終濃度 μg/ml
|
0
|
25
|
125
|
250
|
500
|
750
|
1000
|
1500
|
2000
|
|
總體(tǐ)積 μl
|
100 μl
|
3.
将适當體(tǐ)積的待測樣品加入到試管中，并用PBS補足到100
μl；
4.
向試管中加入2ml
BCA工(gōng)作(zuò)液，混勻，37℃放(fàng)置30分(fēn)鍾；
注：也可(kě)以室溫放(fàng)置2小時，或60℃放(fàng)置30分(fēn)鍾。BCA法測定蛋白(bái)濃度時，顔色會随着時間的延長不斷加深。并且顯色反應會因溫度升高而加快(kuài)。如(rú)果濃度較低，适合在較高溫度孵育，或适當延長孵育時間。
5.
測定562
nm 處的吸光(guāng)值，并記錄讀(dú)數；以不含BSA
的樣品的光(guāng)吸收值作(zuò)爲空白(bái)對照(zhào)。
6. 以A562爲縱坐(zuò)标，BSA含量爲橫坐(zuò)标，繪制标準曲線，計(jì)算樣品中的蛋白(bái)濃度。如(rú)果所得(de)到的蛋白(bái)濃度不在标準曲線範圍内，請(qǐng)稀釋樣品後重新測定。
● References：
1.
Smith,
P.K., et al. (1985). Measurement of protein using bicinchoninic acid. Anal.
Biochem. 150:76-85.
2.
Wiechelman,
K., et al. (1988). Investigation of the bicinchoninic acid protein assay:
Identification of the groups responsible for color formation. Anal Biochem. 175:231-7.
3.
Kessler,
R. and Fanestil, D. (1986). Interference by lipids in the determination of
protein using bicinchoninic acid. Anal. Biochem. 159:138-42.
4.
Brown,
R., et al. (1989). Protein measurement using bicinchoninic acid:
elimination of interfering substances. Anal. Biochem. 180:136-9.
BCA蛋白(bái)定量試劑盒
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