Phospho-AP2M1 (Thr156) Antibody [J20G9]

Experimental Protocol:

 

Sample Preparation

1. Tissue samples: Disrupt the tissue, add an appropriate amount of preheated Hot 1% SDS Lysis Buffer (containing Protease Inhibitor Cocktail), and homogenize at 90 - 95℃.

2. Adherent cell samples: Aspirate the culture medium and wash the cells twice with ice-cold PBS. Add an appropriate amount of preheated Hot 1% SDS Lysis Buffer (containing Protease Inhibitor Cocktail), perform thermal lysis at 90 - 95℃ for 10 minutes, and repeatedly pipette to resuspend the cells during this period to ensure full contact between the cells and the hot lysis buffer.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice.Add an appropriate amount of preheated Hot 1% SDS Lysis Buffer (containing Protease Inhibitor Cocktail), perform thermal lysis at 90 - 95℃ for 10 minutes, and repeatedly pipette to resuspend the cells during this period to ensure full contact between the cells and the hot lysis buffer.

4. Transfer the obtained homogenate/lysate to a centrifuge and centrifuge for 15 min, then collect the supernatant;

5. Take a small amount of the lysate to determine the protein concentration;

6. Add protein loading buffer, heat 20 μL of the sample at 95~100°C for 5 min, let it cool down on ice and then centrifuge for 5 min.

 

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

 

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

 

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution ( recommending 5% BSA solution) for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

 

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:10000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

 

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.