BCA蛋白濃度測定試劑盒（目錄號：RTP7102）
BCA Protein Assay Kit
● 試劑盒內(nèi)容及保存：
|
貨號
|
產(chǎn)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(biāo)準(zhǔn)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(biāo)準(zhǔn)溶液-20℃貯存；PBS溶液4℃貯存。本試劑盒有效期1年。
● 產(chǎn)品簡介：
BCA蛋白濃度測定試劑盒的原理是蛋白質(zhì)分子中肽鍵結(jié)構(gòu)在堿性環(huán)境下能與Cu2+生產(chǎn)絡(luò)合物，并將Cu2+還原成Cu+，而BCA試劑可以特異性地與Cu+結(jié)合，形成穩(wěn)定的有顏色的復(fù)合物，并在562nm處有最大的光吸收值，該復(fù)合物顏色的深淺與蛋白質(zhì)濃度成正比，可以根據(jù)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(chǎn)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(nèi)有較好的線性關(guān)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(yōu)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(yīng)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(biāo)準(zhǔn)品配制：室溫完全溶解蛋白標(biāo)準(zhǔn)品，取20μl
5mg/ml BSA蛋白標(biāo)準(zhǔn)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(biāo)準(zhǔn)測定溶液：
|
編號
|
0
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
|
|
1 mg/ml BSA 標(biāo)準(zhǔn)溶液 μl
|
5 mg/ml BSA 標(biāo)準(zhǔn)溶液 μl
|
|
BSA標(biāo)準(zhǔn)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(dāng)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(yīng)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(dāng)延長孵育時間。
5.
測定562
nm 處的吸光值，并記錄讀數(shù)；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(biāo)，BSA含量為橫坐標(biāo)，繪制標(biāo)準(zhǔn)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(biāo)準(zhǔn)曲線范圍內(nèi)，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(biāo)準(zhǔn)品配制：室溫完全溶解蛋白標(biāo)準(zhǔn)品，取150μl
5mg/ml BSA蛋白標(biāo)準(zhǔn)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(biāo)準(zhǔn)測定溶液：
|
編號
|
0
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
|
|
1 mg/ml BSA 標(biāo)準(zhǔn)溶液 μl
|
5 mg/ml BSA 標(biāo)準(zhǔn)溶液 μl
|
|
BSA標(biāo)準(zhǔn)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(dāng)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(yīng)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(dāng)延長孵育時間。
5.
測定562
nm 處的吸光值，并記錄讀數(shù)；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(biāo)，BSA含量為橫坐標(biāo)，繪制標(biāo)準(zhǔn)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(biāo)準(zhǔn)曲線范圍內(nèi)，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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