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      Oriscience Supersensitive ECL Kit 是一種基于辣根過氧化物酶(horseradish peroxidase, HRP)的極敏型ECL化學(xué)發(fā)光試劑盒。本試劑盒對(duì)化學(xué)發(fā)光液成分進(jìn)行了優(yōu)化，應(yīng)用了不同于常見的魯米諾-H₂O₂體系的新型的增強(qiáng)劑和氧化劑，具有靈敏度極高，發(fā)光迅速，亮度強(qiáng)，穩(wěn)定性好等特點(diǎn)，可使用較高的抗體稀釋倍數(shù)（1:5000~1:10000），因此在很大程度上節(jié)約抗體，且試劑中不含有毒成分，實(shí)驗(yàn)安全性更高。 

產(chǎn)品特色

靈敏度更高

發(fā)光迅速，曝光時(shí)間更短

穩(wěn)定性更好，發(fā)光信號(hào)持久

節(jié)約抗體，背景更低

安全性高

儲(chǔ)存條件

4℃避光保存

數(shù)據(jù)展示

Oriscience Supersensitive ECL與某進(jìn)口品牌超敏ECL對(duì)比圖

不同量的Hela細(xì)胞裂解液的Anti-actin Western Blotting結(jié)果（一抗稀釋倍數(shù) 1：10000，二抗稀釋倍數(shù)：1：10000）。除使用的ECL底物不同而外，其余實(shí)驗(yàn)條件完全一致。

注意事項(xiàng)

1. 在配置ECL工作液過程中吸取A液和B液時(shí)應(yīng)避免交叉污染。

2. ECL工作液應(yīng)在每次使用前新鮮配制，避免室溫放置時(shí)間過長。

3. 曝光時(shí)間過長或蛋白過量，將會(huì)導(dǎo)致過曝，使條帶強(qiáng)弱變化失去線性關(guān)系；曝光不足則條帶模糊。

4. ECL發(fā)光液是HRP的顯色底物，因此檢測對(duì)象必須由HRP標(biāo)記。

5. 本產(chǎn)品僅供科研使用。請(qǐng)勿用于醫(yī)藥、臨床診斷或治療，食品及化妝品等用途。

Q1：背景臟，信噪比低，出現(xiàn)非特異性條帶

a. 抗體特異性低：選用特異性更高的抗體。

b. 顯影時(shí)間過長：在確保獲得足夠強(qiáng)度的目標(biāo)蛋白信號(hào)的前提下，縮短顯影時(shí)間。

c. 抗體孵育和洗膜過程中的污染：確保操作過程中所有試劑和用具的潔凈度。

d. 未進(jìn)行非特異性封閉或封閉不充分：延長封閉時(shí)間，考慮更換合適的封閉劑。

e. 一抗?jié)舛冗^高導(dǎo)致非特異性結(jié)合：稀釋抗體至合適濃度，以更高稀釋度抗體孵育更長時(shí)間（耗時(shí)長但特異性結(jié)合最好）。

f. 二抗與封閉劑非特異性結(jié)合：設(shè)置二抗對(duì)照(不加一抗)以明確，如確實(shí)存在此種情況，可在孵育和洗滌液中加入溫和去污劑，如吐溫20。

OriNote：脫脂奶粉含有酪蛋白，對(duì)于酪蛋白激酶，該蛋白本身就是一種磷酸化蛋白底物，有可能會(huì)結(jié)合磷酸化特異性抗體而易產(chǎn)生高背景。使用 BSA 代替奶粉作為封閉劑。

g. 膜干燥，導(dǎo)致高背景： 在孵育過程中防止膜變干，在任何步驟都保證膜有充分的反應(yīng)液。

h. 未結(jié)合蛋白質(zhì)洗滌不充分：增加洗滌次數(shù)。

Q2：目的條帶過曝

a. 減少蛋白上樣量。

b. 縮短曝光時(shí)間。

c. 用緩沖液適當(dāng)洗滌后重新顯影。

d. 滴加ECL后放置一段時(shí)間再顯影。

Q3：目的條帶弱

a. 轉(zhuǎn)膜不充分：優(yōu)化轉(zhuǎn)膜條件，提高轉(zhuǎn)膜效率。

b. 增加蛋白上樣量。

c. 抗體稀釋倍數(shù)過大：降低抗體稀釋倍數(shù)以提高其濃度。

d. 抗體使用時(shí)間過長導(dǎo)致抗體失效：重新配制抗體。

Q4：背景有不均勻的白色斑點(diǎn)

a. 轉(zhuǎn)膜時(shí)膜上有氣泡或抗體在膜上分布不均：轉(zhuǎn)膜過程中盡量去除氣泡，抗體孵育時(shí)保持搖動(dòng)。

Q5：背景有黑色斑點(diǎn)

a. 抗體結(jié)合了封閉劑：過濾封閉劑。

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