太鼎生物科技/太鼎食安科技

蛋白凝膠銀染試劑盒 | Protein Silver Stain Kit Servicebio貨號G2080-25T

產品介紹:

銀染(Silver staining)是一種高靈敏度的染色方法，其基本原理是通過還原陰離子(reduced anion)在蛋白質上形成黑色沉澱(black precipitate)，從而顯示蛋白質條帶。銀染(silver stain)比傳統的考馬斯亮藍染色靈敏 100 倍，可檢測小於 0.5 ng 的蛋白質。由於其高靈敏度，銀染(silver stain)被廣泛用於 2D 凝膠分析(2D gel analysis)和垂直 PAGE 凝膠(vertical PAGE gel)中，用於極低蛋白質含量的測定。它是檢測凝膠中low abundance proteins的常用方法。本產品可用於SDS-PAGE或PAGE聚丙烯酰胺凝膠中的蛋白質檢測，靈敏度高，操作方便，背景幾乎無色，一塊凝膠的銀染可在90分鐘內完成。本試劑盒可對25塊相同規格的凝膠進行染色。

Assay Protocol

1、固定(Fixation)：配製固定液(如下表)。將凝膠置於乾淨的染缸中，用超純水(ultra-pure water)清洗3次，加入20 mL固定液(fixing solution)覆蓋凝膠，用水平搖床(horizontal shaker)（60-70 rpm）緩慢搖動，孵育30分鐘。為防止醋酸(acetic acid)和乙醇(ethanol)揮發，請用保鮮膜密封染缸。延長固定時間可以進一步降低背景。

2、洗滌(Washing)：倒出固定液(fixing solution)，更換超純水(ultra-pure water)覆蓋膠，水平搖床搖晃3次，每次10 min。

3、敏化(Sensitization)：按下表配製敏化工作液。加入敏化液(sensitization solution)覆蓋凝膠，凝膠在水平搖床上緩慢搖動孵育1分鐘。

4、洗滌(Washing)：倒出敏化工作液(sensitization working solution)，換上超純水，搖動搖床3次，每次20 s。

5、染銀(Silver dyeing)：配製染銀液(如下表)。加入銀染液(Silver staining solution覆蓋凝膠，凝膠緩慢震盪孵育20分鐘。銀染液(Silver dyeing solution)應即配即用，盡量在2小時內用完。

6、水洗(Wash with water)：棄去染液(dye solution)，換上超純水，搖動搖床3次，每次20 s。

7、顯色(Color development)：按下表配製顯色液(developing solution)。加入顯影液(developing solution) 覆蓋凝膠，將凝膠震盪孵育1-5分鐘，直至出現理想的目的蛋白條帶。

9、終止(Termination)：準備開發termination solution (如下表) 。加入顯影終止液(development stop solution)覆蓋凝膠，用搖床孵育凝膠2分鐘。過程中產生氣體屬於正常現象，產生的氣體為CO2。然後棄去終止液，用超純水洗滌凝膠2-5分鐘。

9、存放(Storage)：清洗乾淨後，可放置凝膠進行觀察和拍照。染色後的凝膠可以保存在超純水或1%醋酸溶液中，或適當乾燥。

Note:

1. To prevent interference from impurities, use high purity water (resistance > 16 MΩ/cm) and clean glassware or plastic containers. Do not use metal containers.
2. Please take protective measures during the whole experiment. Ultrapure water, absolute ethanol and glacial acetic acid should be prepared by yourself.
3. Do not directly touch or press the colloid during the operation, and the dye VAT used for dyeing cannot use metal products, glass or plastic containers are the best.
4. Keep the sensitizer at room temperature away from light. Do not store it at 4℃, otherwise it will fail.
5. All working fluids shall be prepared and used at present. After opening and using the reagent, it is necessary to tighten the cap of the bottle to prevent the volatilization of the solution and chemical reaction with substances in the air, which will affect the dyeing effect.
6. The developing solution is stored at 4℃for a long time with crystal precipitation (normal phenomenon), and can be incubated at 37℃ until completely dissolved before use.
7. When the gel is thick or the operating temperature is low, the color development time can be extended appropriately.
8. The dyeing time, washing time, color development and termination time of each step should be well controlled, otherwise the glue background will become dark and it is not easy to observe the target band.

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