BCA蛋白濃度測定試劑盒（目錄號：RTP7102）
BCA Protein Assay Kit
● 試劑盒內容及保存：
|
貨號
|
產品名稱
|
包裝
|
貯存
|
|
RTP7102-01
|
BCA試劑A
|
100 ml
|
RT
|
|
RTP7102-02
|
BCA試劑B
|
3 ml
|
RT
|
|
BSA-01
|
牛血清白蛋白(BSA)標準溶液（5 mg/ml）
|
2×1 ml
|
-20℃
|
|
RT0280-02
|
PBS溶液
|
10 ml
|
4℃
|
|
|
說明書
|
1份
|
|
● 儲存條件和效期：
試劑A和試劑B室溫貯存；牛血清白蛋白標準溶液-20℃貯存；PBS溶液4℃貯存。本試劑盒有效期1年。
● 產品簡介：
BCA蛋白濃度測定試劑盒的原理是蛋白質分子中肽鍵結構在堿性環境下能與Cu2+生產絡合物，并將Cu2+還原成Cu+，而BCA試劑可以特異性地與Cu+結合，形成穩定的有顏色的復合物，并在562nm處有最大的光吸收值，該復合物顏色的深淺與蛋白質濃度成正比，可以根據吸收值的大小來測定蛋白的含量。
本試劑盒可以檢測500個樣品（使用微孔板）或50個樣品（使用試管）。
● 產品特點：
1. 靈敏度高，檢測濃度下限達到25μg/ml（在20-1000μg/ml濃度范圍內有較好的線性關系），最小檢測蛋白量達到0.2μg，待測樣品體積為1-20μl。
2. BCA法測定蛋白濃度的最大優點是蛋白濃度的測定可以耐受高濃度的去垢劑，可以兼容樣品中高達5%的SDS，5%的Triton
X-100，5%的Tween
20, 60, 80。但受螯合劑和略高濃度的還原劑的影響，需確保EDTA低于10mM，無EGTA，二硫蘇糖醇低于1mM，β-巰基乙醇低于0.01%。
● 操作方法
BCA工作液配制：
將試劑A和試劑B按照體積比50:1比例混合，配成BCA工作液。
如，取50ml 試劑A與1ml試劑B混合，配成51 ml BCA工作液。兩者混合時會有沉淀形成，徹底混勻后沉淀消失，溶液應為澄清淡藍色溶液。
注：BCA工作液室溫可放置一周不失效。
微孔板測定程序：（工作范圍20-2000 μg/ml）
1. 蛋白標準品配制：室溫完全溶解蛋白標準品，取20μl
5mg/ml BSA蛋白標準溶液用PBS溶液稀釋至100μl，使其終濃度為1.0 mg/ml。
2.
按照下表配制BSA標準測定溶液：
|
編號
|
0
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
|
|
1 mg/ml BSA 標準溶液 μl
|
5 mg/ml BSA 標準溶液 μl
|
|
BSA標準溶液 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
|
|
總體積 μl
|
20 μl
|
3.
將適當體積的待測樣品加入到微孔板中，并用PBS補足到20
μl
4.
向微孔板中加入200
μl BCA工作液，混勻，37℃放置30分鐘；
注：也可以室溫放置2小時，或60℃放置30分鐘。BCA法測定蛋白濃度時，顏色會隨著時間的延長不斷加深。并且顯色反應會因溫度升高而加快。如果濃度較低，適合在較高溫度孵育，或適當延長孵育時間。
5.
測定562
nm 處的吸光值，并記錄讀數；以不含BSA
的樣品的光吸收值作為空白對照。
6. 以A562為縱坐標，BSA含量為橫坐標，繪制標準曲線，計算樣品中的蛋白濃度。如果所得到的蛋白濃度不在標準曲線范圍內，請稀釋樣品后重新測定。
試管測定程序：（工作范圍20-1000
μg/ml）
1. 蛋白標準品配制：室溫完全溶解蛋白標準品，取150μl
5mg/ml BSA蛋白標準溶液，加入600μl PBS溶液稀釋至750μl，使其終濃度為1.0 mg/ml。
2.
按照下表配制BSA標準測定溶液：
|
編號
|
0
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
|
|
1 mg/ml BSA 標準溶液 μl
|
5 mg/ml BSA 標準溶液 μl
|
|
BSA標準溶液 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
|
|
總體積 μl
|
100 μl
|
3.
將適當體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工作液，混勻，37℃放置30分鐘；
注：也可以室溫放置2小時，或60℃放置30分鐘。BCA法測定蛋白濃度時，顏色會隨著時間的延長不斷加深。并且顯色反應會因溫度升高而加快。如果濃度較低，適合在較高溫度孵育，或適當延長孵育時間。
5.
測定562
nm 處的吸光值，并記錄讀數；以不含BSA
的樣品的光吸收值作為空白對照。
6. 以A562為縱坐標，BSA含量為橫坐標，繪制標準曲線，計算樣品中的蛋白濃度。如果所得到的蛋白濃度不在標準曲線范圍內，請稀釋樣品后重新測定。
● 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.
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