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This kit provides a simple and fast 6% SDS-PAGE color (green) gel preparation method. Users only need to prepare their own gel-making equipment to prepare the gel, which greatly simplifies the gel-making process. The upper gel (concentrated gel) reagent provided in the kit can prepare a colored (green) upper gel. The green staining kit can stably exist in the upper gel (concentrated gel, accumulated gel), and will not enter the lower gel with electrophoresis ( Separating gel), so the addition of the green dye can make the upper gel protein spotting hole clearly appear, it is easy to judge the margin of the sample hole or whether it is distorted or damaged, and it is convenient to add the protein sample to the spotting hole accurately. The staining kit will not affect the electrophoresis and staining effect. After the electrophoresis is completed, it is easy to identify and remove the upper layer gel, and does not affect subsequent Western Blot and other experiments. The specific number of gel blocks is related to the thickness of the gel and the size of the gel.
Storage and transportation
Stored at 4°C and away from light, valid for one year; after AP is dissolved, it is stored in small tubes at -20°C, and valid for one year.
|Upper gel solution A||50mL|
|Upper gel solution B (green)||50mL|
|6% lower gel solution A||125mL|
|6% lower gel solution B||125mL|
|AP (Ammonium Persulfate)||1g(Powder)|
According to the molecular weight of the target protein, select the appropriate concentration of SDS-PAGE lower gel (separation gel), please refer to the following table:
|SDS-PAGE separation gel concentration||Best separation range (kDa)|
2. Take an appropriate amount of AP powder and prepare a 10% AP solution. (For example, take 180 mg of AP powder and add 1.8 mL of pure water to dissolve the 10% AP solution.) The AP solution is prone to failure. The prepared 10% AP solution can be stored at 4°C for 1-2 weeks, and the remaining AP powder can be stored at -20°C.
3.According to the experimental requirements, mix solution A and B in equal proportions, add an appropriate amount of 10% AP solution, and prepare the lower gel solution and the upper layer gel solution respectively. For glass plates of different specifications and different thicknesses, the preparation volume of the upper gel and the lower gel solution can be adjusted in equal proportions. Taking the commonly used 8.3 cm×7.3 cm gel plate as an example, the recommended preparation system is as follows:
|Group||Component||Single piece of gel (1.0 mm thickness)||Two pieces of gel (1.0 mm thickness)|
Lower gel solution
|6% lower gel solution A||2.5mL||5mL|
|6% lower gel solution B||2.5mL||5mL|
|10% AP solution||50μL||100μL|
Upper gel solution
|Upper gel solution A||1mL||2mL|
|Upper gel solution B (green)||1mL||2mL|
|10% AP solution||50μL||40μL|
4. Prepare the gel tool and pour the prepared lower layer gel solution, and immediately add the prepared upper layer gel solution slowly and evenly (distilled water can also be used to seal the lower layer gel liquid surface, 15-30 minutes later until the lower layer gel solidifies Add the prepared upper layer gel solution after it is full), insert the comb, and wait for it to solidify (about 15-30 min) before use.
5. If the prepared gel cannot be used on the same day, it can be stored at 4°C for 1-2 days before use.
1. There is monomer acrylamde in the premix, which is harmful to the human body. Please take protective measures during operation.
2. In order to ensure that the sample enters the separation gel at the same time, when directly pouring the top gel, the prepared top gel solution should be added slowly and evenly.
3. Pour the gel as soon as possible after adding AP, do not leave it for a long time.
4. The solidification of the gel has a significant positive correlation with the temperature. Under the same conditions, the higher the temperature, the faster the solidification rate.
5. This product has been added with an appropriate amount of TEMED substitutes. If you need to further accelerate the gel speed, you can add an appropriate amount of TEMED as needed before the gel is prepared, and at the same time increase the AP usage by 0.5 times.