Gel Electrophoresis

1. Extraction of total tissue protein: 2.1 Wash the tissue 1-2 times with pre-cooled PBS, cut into small pieces and place in a grinding tube, add 3pcs, 3mm grinding beads, and add 10 times the tissue volume of lysis solution (for example: 100mg of tissue, add 1000ul of lysis solution liquid), set the grinding program for tissue grinding; 2.2 Take out the grinding tube after grinding and place it on ice or in fourth-degree lysis solution for 30 minutes; 2.3 Centrifuge at 12000 rpm, 4°C for 10 minutes, collect the supernatant, which is the total protein solution. 2. Protein concentration determination (optional): Determine the protein concentration as needed, take the undenatured protein solution, and use the BCA protein concentration determination kit to measure the protein concentration. For specific methods, refer to the kit instructions; 3. Protein denaturation:Add 5* reduced protein loading buffer to the protein solution at a ratio of 4:1, denature it in a metal bath at 95°C for 10 minutes, and store it in a -20°C or -80° refrigerator for later use; 4. Electrophoresis 4.1 Clean the glass plate; 4.2 Gel preparation and sample loading; 4.2.1 ①. First, move the clamping plates on both sides to the bottom, completely open the glands on both sides, insert the concave glass and flat glass from the top diagonally and place them to the bottom. The upper part of the glass is stuck in the slots on both sides; ②. Turn up the glands on both sides, pinch the left part of the gland with your hands at the same time, pull the left clamping plate upward, and lock it to the top; then pinch the right side of the gland at the same time, pull the right clamping plate upward, and lock it. to the highest; ③. After confirming that the electrophoresis glass is clamped and aligned, unscrew the knobs on both sides of the gel making base, then place the electrophoresis bracket into the middle of the gel making base and clamp it, then press the main body bracket with your hands and tighten the knobs on both sides until Rotate to the limit. 4.2.2 Select gel making kits of different concentrations according to experimental requirements, mix solutions A and B in equal proportions, and prepare lower gel solution and upper gel solution respectively. For glass plates of different specifications and thicknesses, the volumes of the upper glue and lower glue solutions can be adjusted in equal proportions. Taking the commonly used 8.3 cm × 7.3 cm gel plate (single piece) as an example, the recommended preparation system is as follows:; Formulation group Formulation 0.75 mm Glass Plate 1.0 mm Glass Plate 1.5 mm Glass Plate Lower glue solution 10% Lower glue solution A 2 mL 2.5 mL 4 mL 10% Lower glue solution B 2 mL 2.5 mL 4 mL AP 24 μL 30 μL 48 μL Upper glue solution Upper glue solution A 1 mL 1 mL 1.5 mL Upper glue solution B(Red Color) 1 mL 1 mL 1.5 mL AP 12 μL 12 μL 18 μL 4.2.3 After assembling the glue maker, first add the prepared lower glue solution, and then use pure water or ethanol to seal the lower glue surface. After the lower glue has fully solidified (about 10-15 minutes), discard the water or ethanol, and use filter paper to absorb the remaining liquid, then add the prepared upper glue solution, insert the comb, and wait for it to solidify (about 10-15 minutes) before use; 4.2.4 Remove the main body of the gel maker, carefully pull out the comb, and prepare to start electrophoresis; 5. After placing the main body of the gel maker into the electrophoresis tank, fill the inside with electrophoresis buffer and add 1/3 with the outside. Use a constant voltage of 200V for 30 minutes until the bromophenol blue is approximately 1cm away from the bottom. The electrophoresis is terminated and the electrophoresis is ready for transfer. 6. Transfer film 6.1 Prepare 6 pieces of 7×9cm transfer filter paper (thin) and 5×8cm PVDF membrane. The PVDF membrane must be activated with ethanol for 2 minutes before use; 6.2 Put the transfer clip, two sponges, filter paper and activated PVDF membrane in the container with transfer solution; 6.3 Spread the transfer folder, with red on the left and black on the right. Add a sponge and three pieces of filter paper to each side; 6.4 Carefully peel off the separation glue and place it on the filter paper (the glue is placed on the side of the black transfer clip). Use the transfer liquid to rinse the bubbles on the glue. Slowly stick the PVDF film to the glue. Be careful not to have any bubbles. Then stick the transfer film in turn. Membrane filter paper, transfer sponge; 6.5 Transfer conditions (wet transfer): Constant current, 300mA for half an hour. 7. Immune response 7.1 Place the transferred membrane into an incubation box containing TBST, rinse it quickly, then add 5ml of 5% skimmed milk, place it on a decolorizing shaker, and block at room temperature for 30 minutes; 7.2 According to the antibody instructions, dilute the primary antibody. After configuration, pour out the blocking solution in the incubation box, add the prepared primary antibody, and incubate at 4°C on a shaker overnight (shake the shaker slowly); 7.3 Recover the primary antibody, rinse the membrane quickly with TBST three times, then add TBST, place it on a destaining shaker for rapid elution, wash three times for 5 minutes each time; 7.4 Dilute the secondary antibody with TBST at a ratio of 1:5000, then add it to the incubation box, place it on a shaker and shake slowly, and incubate at room temperature for 30 minutes; 7.5 Rinse the membrane quickly with TBST three times, then add TBST, place it on a destaining shaker for rapid elution, wash three times for 5 minutes each time. 8. ChemiluminescenceMix ECL A and B solutions in a ratio of 1:1 and set aside. Take out the eluted PVDF membrane and place it on absorbent paper. Slightly absorb the liquid on the membrane and put the membrane into the mixed ECL. In the luminescent liquid, let the liquid completely immerse the membrane. After the reaction for 1 minute, take out the membrane and place it on the chemiluminescence instrument tray. Start chemiluminescence according to the preset program. After the exposure is completed, save the original image in TIFF format. 9. WB results and analysis 

Introduction Nucleic acid electrophoresis is a fundamental technique in molecular biology used to separate DNA or RNA fragments based on their size. This method relies on an electric field to move negatively charged nucleic acids through a gel matrix, typically agarose or polyacrylamide. The separated fragments can then be visualized and documented using a gel documentation system, such as Yanbiotech’s YB-GD1, for accurate analysis. Below is a detailed step-by-step protocol for performing nucleic acid electrophoresis. Materials Required 1. Gel preparation: Agarose powder (for agarose gel electrophoresis) TAE (Tris-Acetate-EDTA) or TBE (Tris-Borate-EDTA) buffer Gel casting tray and comb Ethidium bromide (EB) or safer alternatives like SerRed. 2. Sample preparation: DNA/RNA samples Loading dye DNA ladder (size standard) 3. Electrophoresis setup: Electrophoresis chamber Power supply Yanbiotech YB-GD1 Gel Documentation System (or UV transilluminator for visualization) Step-by-Step Procedure 1. Preparing the Agarose Gel 1.1: Prepare the buffer (usually 1X TAE or TBE). 1.2: Weigh the appropriate amount of agarose (e.g., 1% agarose for standard DNA separation). 1.3: Mix agarose with buffer and heat until fully dissolved (microwave for 30-60 sec). 1.4: Cool the solution slightly (~60°C), then add DNA stain (e.g., EB or SerRed). 1.5: Pour the gel into a casting tray with a comb inserted and let it solidify (~20-30 min). 2. Loading the Samples 2.1: Carefully remove the comb and place the gel in the electrophoresis chamber. 2.2: Fill the chamber with enough buffer to cover the gel. 2.3: Mix DNA samples with loading dye (for tracking migration). 2.4: Load the samples into the wells, including a DNA ladder for size reference. 3. Running the Electrophoresis 3.1: Connect the electrodes (negative to the sample side, positive to the opposite end). 3.2: Apply voltage (typically 80-120 V for agarose gels). 3.3: Run until the dye front migrates ¾ of the gel length (~30-60 min, depending on fragment size). 4. Visualizing and Documenting Results with YB-GD1 4.1: Turn off the power supply and carefully remove the gel. 4.2: Place the gel in the Yanbiotech YB-GD1 Gel Documentation System for high-resolution imaging. The YB-GD1 offers UV and white LED excitation, allowing visualization of SerRed, and other common nucleic acid stains. Its high-sensitivity CCD camera ensures clear and precise band detection. The accompanying software enables automatic band analysis and size estimation by comparing samples to the DNA ladder. 4.3: Save and export the gel image for further documentation and publication. Troubleshooting Tips Smearing: May indicate degraded DNA or too much sample loaded. No bands: Check staining method, electrophoresis conditions, or DNA quantity. Uneven bands: Ensure gel was poured evenly and buffer concentration was correct. Poor image quality (if using YB-GD1): Adjust exposure time or focus settings in the software. Conclusion Nucleic acid electrophoresis is a simple yet powerful technique for analyzing DNA and RNA. By following these steps carefully and using Yanbiotech’s YB-GD1 Gel Documentation System, researchers can achieve high-quality visualization, accurate sizing, and professional documentation of nucleic acid fragments. This method is widely used in cloning, PCR verification, and genetic fingerprinting, making it essential for molecular biology laboratories. Why Choose Yanbiotech’s YB-GD1? ✔ High-resolution imaging for precise DNA/RNA band detection ✔ UV and LED options for compatibility with multiple stains ✔ User-friendly software for automatic band analysis ✔ Compact and efficient design, ideal for lab workflows Would you like any further modifications or additional details on the YB-GD1 system?