梯度型快速制胶试剂
传统的梯度SDS-PAGE制备困难，批次间的一致性也难以维持。WB梯度凝胶溶液是专门为解决这些难题而设计的创意产品，并且与您原有的Laemmli系统或蛋白质检测（免疫染色、比色染色）系统100%相容。因此，使用WB梯度凝胶溶液，您可以在一张凝胶上完成更多的测定，重复性更好，分离效率更高。
WB Quickly Gradient gel solution
|
|
|
Introduction Many kinds of SDS-PAGE electrophoresis system are widely used in proteomic studies. Among them,the Tris-glycine buffer system is most popular used for researcher because of its higher resolution and convenience to prepare. However,it is hard to separate both higher and lower molecular weight proteins at the fixed percentage of SDS-PAGE,nor use gradient SDS-PAGE. Hence,the traditional gradient SDS-PAGE is hard to prepared and consistency between lots is also barely to maintain. WB Gradient Gel Solution is a creative product that specially designed for resolving these difficulties and 100% compatible to your original Laemmli system or protein detection (immune-staining,colorimetric staining) system. Therefore,you can complete more assays in one gel with WB Gradient Gel Solution, which is more repeatable and excellent separation efficiency. Reagent 1. WB Gradient Gel Solution 2. WB Gradient Gel sample buffer 3. Provided by user:Acrylamide/Bis,TEMED,APS,S-S bond reducing agent. Protocol For general SDS-PAGE protocol A. Preparation of gel solution 1. According to the following information to prepare the SDS-PAGE solution by gently mix WB Gradient Gel Solution with Acrylamide/Bis-acrylamide solution (Table 1). 2. Gently and mix 20ul of TEMED into 10ml well mixed solution prepared in step 1. 3. Gently and mix 200ul of 10% APS into 10ml well mixed solution prepared in step 2. It is not necessary to prepare stacking gel ! Therefore directly load the gel solution to a full volume of the glass plate. 4. Directly insert clean comb,avoid trapping air bubbles and gel solution leakage. The gel should be polymerized after 15minutes at room temperature. 5. Carefully remove the comb;gently rinse the lane space by distilled water. B.Preparation of sample 1. Add appropriate reducing agent (0.7M B-ME,0.3M DTT,or 0.3M DTE) to Fu Gradient Gel sample buffer before use. 2. Dilute 1 part of WB Gradient Gel sample buffer with 3~5 part of quantified protein sample. According to protein maker ladder, each volume of sample buffer mix should be the same by adding lysis buffer you used. 3. Heating at >90℃ for 5minutes. 4. Directly load into well. For secondary dimension electrophoresis by IEF strip A. Prepare gel solution 1. According to the following information to prepare the SDS-PAGE solution by gently mix WB Gradient Gel Solution with Acrylamide/Bis-acrylamide solution (Table 1). 2. For example, to prepare 2 pieces of 10cm acrylamide gel, please mix 10ml of WB Gradient Gel Solution gel buffer with 2.39ml Acrylamide/Bis-acrylamide solution (it is recommend to use 37.5:1,40% w/v acrylamide/Bis-acrylamide solution). 3. Gently add and mix 20ul of TEMED into 10ml well mixed solution prepared in step 1. 4. Gently add mix 200ul of 10% APS into 10ml well mixed solution prepared in step3. 5. Pour gel solution prepared in step 4. Into plate space, leave enough upper space to insert IEF strip. Add butanol (distilled water or ethanol can also be used) to make a smooth surface. 6. The gel should be polymerized after 15minutes at room temperature. Dry the stop surface of the gel by filter paper. 7. Carefully insert pretreated IEF strip into upper side of the gel. Strips were overlayed with agarose sealing solution (0.5% agarose and 2.5~5% GredientMagic sample buffer prepared with Tris-glycine running buffer). Electrophoresis 1. Starting electrophoresis at constant voltage (~90 to 110V), at this condition, the electric current should be 30-50 mA to each gel. 2. Continue performing electrophoresis until tracking dye is reach to the bottom of the gel. 3. During electrophoresis, remember that WB Gradient Gel sapmle buffer’s tracking bye will forming to a small gradient region, but not stacking to a sharp band such as traditional bromophenol blue based tracking dye. |
   |