Purpose: To separate and analyze proteins based on molecular weight and charge, essential for protein purification, identification, and functional analysis.
a. Materials
Glass plates, gel mold, gel casting rack, pipettes and tips, test tubes, 30% Acrylamide, 4×SDS-PAGE separation gel buffer, 4×SDS-PAGE stacking gel buffer, 10% Ammonium persulfate (APS), TEMED, ddH2O, waste container, comb. b. Procedure:
i. Insert thin glass plate into the thick glass plate to form a container for the gel solution and secure the plates in the casting rack.
ii. 10% Separation Gel Preparation:
1. Add 2.5 ml of 4×SDS-PAGE separation gel buffer to a test tube.
2. Add 4.1 ml of ddH2O.
3. Add 3.3 ml of 30% acrylamide.
4. Add 100 µl of 10% APS.
5. Add 10 µl of TEMED, mix quickly.
iii. Transfer the gel solution into the space between the glass plates using a pipette, then cover with methanol to level the gel.
iv. Let the separation gel solidify for 30 minutes, then discard the methanol.
v. 5% Stacking Gel Preparation:
1.Add 1.25 ml of 4×SDS-PAGE stacking gel buffer to a test tube.
2.Add 2.84 ml of ddH2O.
3.Add 0.83 ml of 30% acrylamide.
4.Add 75 µl of 10% APS.
5. Add 7.5 µl of TEMED, mix quickly.

vi. Transfer the stacking gel solution on top of the separation gel using a pipette, insert the comb, and let it solidify for 30 minutes.
vii. Remove the comb before use.

Purpose:
The aim of this experiment is to detect specific proteins by using antibodies that specifically recognize the target proteins. It is also used to measure the expression levels of these proteins by comparing the intensity of the bands in different samples, assess the molecular weight of the proteins by comparing them to standard proteins, and evaluate the modification status of proteins, such as phosphorylation or glycosylation, by analyzing changes in their migration under different conditions. a. Principles:
i. SDS-PAGE: Different proteins carry different charges and molecular weights, so after running the gel, proteins will be distributed across different positions on the gel. ii. Primary antibodies bind to the target protein, and secondary antibodies bind to the primary antibody. Detection of the emitted signal allows visualization of the protein’s position and expression levels.
b. Procedure:
i. Gel Electrophoresis (Running the Gel)
Prepare the electrophoresis buffer: mix 500 ml deionized water with SDS buffer powder.
Assemble the gel in the electrophoresis tank (red corresponds to the positive electrode, blue to the negative). Pour electrophoresis buffer into the tank until it reaches the marked line.
Load 20 μL of each protein sample into the wells of the gel (repeat until all samples are loaded).
Load 15 μL of sample loading buffer mixed with 5 μL of protein marker into a separate well.
Connect the power supply and run the gel for 40 minutes, or until the marker reaches the desired position.
ii. Membrane Transfer
Soak two filter papers and two black sponge pads in transfer buffer, and soak one PVDF membrane in methanol.
Remove the gel from the electrophoresis plate, cut the gel based on the protein bands.
Assemble the transfer stack in the following order: black clamp, one black sponge pad, one filter paper, PVDF membrane, the gel, another filter paper, another black sponge pad, and finally, another black clamp. Insert the assembly into the transfer apparatus.
Pour methanol and transfer buffer into the tank until it reaches the marked line for 4 gels.
Transfer the proteins for 1 hour and 40 minutes.

vi. Signal Detection:
For ECL Substrate (Pumino):
Mix ECL A solution and B solution in a 1:1 ratio.
b) Place the PVDF membrane on the detection equipment and evenly coat with the mixed solution.
c) Activate the equipment, calculate the exposure time, and wait for 3 minutes.
d) Observe the image.

For Super Signal Substrate:
a) Mix Super Signal A solution and B solution in a 1:1 ratio.
b) Place the PVDF membrane on the detection equipment and coat with the mixed solution.
c) Activate the equipment, calculate the exposure time, and wait for 42 seconds.
d) Observe the image.

Purpose: Subculturing is a routine method for maintaining cells in culture and is essential for most cell biology experiments. When cells grow to confluence in a flask, they must be diluted and replated in new flasks to continue growing. This process is called subculturing. It is important to maintain strict sterile conditions throughout the process, as any contamination could negatively affect the cells. Suspension cells can be directly transferred, while adherent cells must be digested before subculturing.
a. Medium Change i. Remove the old culture medium. ii. Wash the cells. iii. Add fresh culture medium. iv. Return the flask to the incubator, set the temperature (e.g., 37°C), gas environment (95% sterile air and 5% CO2), and humidity.
b. Subculturing Cells i. Preparation: Prepare the culture medium and necessary reagents (e.g., trypsin, PBS). Ensure all equipment and reagents are sterilized. ii. Observe the cell state: Use a microscope to check the cells' condition, ensuring normal growth and checking for any deformation or contamination (Esophageal epithelial cells should appear elongated). iii. Remove or pour out the old culture medium from the flask. iv. Add PBS rinse solution and gently shake to remove residual serum and dead cells (rinse 3–4 times). v. Add trypsin to cover the cells completely and shake the flask gently. vi. Add fetal bovine serum to stop the digestion reaction. vii. Transfer the cell-containing liquid to a 5 ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes (ensure the centrifuge is balanced). viii. Remove the supernatant, add an appropriate amount of fresh culture medium, and resuspend the cells. ix. Split the resuspended cells and 5 µl of medium into two new culture flasks. Shake horizontally 15–20 times and vertically 15–20 times, then return the flasks to the incubator.

Purpose: PCR is a widely used molecular biology technique, primarily for the rapid and specific amplification of a particular DNA fragment in vitro.1. Amplify specific DNA sequences exponentially to obtain sufficient amounts for subsequent analysis.2. Clone genes or DNA fragments into vectors for gene function studies or protein expression. 3. Detect mutations or polymorphisms in genomes, aiding clinical diagnosis and genetic studies.4. Through reverse transcription PCR (RT-PCR), analyze mRNA expression levels to study gene expression under different conditions.
a. Preparing Reaction Components i. Buffer: Standard buffer contains 1 pmol/ml Tris ·HCl, pH 8.3-9.0 at room temperature, and near pH 7.2 at the extension temperature (72°C). It includes a divalent cation, usually Mg²⁺, to activate the active site of DNA polymerase.
ii. dNTP:Deoxynucleotide triphosphates (dATP, dGTP, dTTP, dCTP) are the building blocks for synthesizing new DNA strands. Their total concentration is typically 200 pmol/ml (saturation level). dNTPs bind to Mg²⁺, reducing free Mg²⁺ concentration, which can affect DNA polymerase activity.
iii. Primers: Short single-stranded DNA fragments that bind to complementary regions on the template DNA, providing a starting point for DNA polymerase to begin synthesizing the new DNA strand. Primers are usually 18-30 bp long. Shorter primers reduce specificity, while longer ones may cause non-specific binding or primer-dimer formation, increasing synthesis costs.
iv. Template: A single-stranded or double-stranded DNA template provides the original sequence for PCR amplification. The template’s purity requirements are low, but it should not contain proteases, nucleases, DNA polymerase inhibitors, or DNA-binding proteins. Template DNA should be stored at low temperatures.
v. Taq DNA Polymerase: DNA polymerase synthesizes DNA from the 5’ to 3’ direction, using the template and primers as a guide. It must be stored at -20°C and contains glycerol as a preservative. The optimal reaction temperature is 72°C (extension temperature). Typically, Taq is added last when preparing the PCR reaction.
b. Mixing Reaction Components: i. Using a pipette, add 11.5 µl of ddH2O (in two steps) into a centrifuge tube. ii. Add 1 µl of primers. iii. Add 2 µl of PCR buffer. iv. Add 2 µl of dNTP. v. Add 2 µl of the template (cDNA). vi. Finally, add 0.5 µl of Taq DNA polymerase.
c. Setting Up the PCR Program: i. Denaturation: Heat the reaction to 94-98°C for 30 seconds to 1 minute, causing the DNA double helix to denature into single strands.
ii. Annealing: Lower the temperature to 50-65°C for 30 seconds to 1 minute to allow primers to bind to the single-stranded DNA template.
iii. Extension: Raise the temperature to 72°C, where DNA polymerase synthesizes a new DNA strand by extending the primer along the template. Steps i-iii are usually repeated for 20-40 cycles to achieve exponential amplification of the DNA fragment. After the final cycle, hold the reaction at 72°C for a final extension of any incomplete fragments, then cool the reaction to 4°C.
Notes: Ensure the concentration of each component is appropriate to avoid inhibiting the PCR reaction. Primer design must be specific to prevent dimer formation or non-specific amplification. Extraction and purification of the PCR product may be critical for subsequent analyses.