JoVE 実験百科事典
微生物学
0 回視聴 • 2:58 分 • July 1st, 2026
Take an SDS-PAGE gel containing resolved proteins from lysates of cells expressing CaeB, an anti-apoptotic bacterial protein, and from control cells lacking CaeB.
Both cell types carry an antibiotic-inducible expression system and were treated with the antibiotic to induce expression of a pro-apoptotic protein.
In control cells, this triggers apoptotic signaling, activating an apoptotic marker.
In CaeB-expressing cells, CaeB inhibits the apoptotic signaling, reducing apoptotic marker activation.
Using an electric field, transfer proteins from the gel onto a membrane.
Add a blocking solution to reduce non-specific antibody binding.
Incubate with a primary antibody targeting the activated apoptotic marker.
Wash to remove unbound antibody.
Incubate with an enzyme-conjugated secondary antibody targeting the primary antibody. Wash again, and add a chemiluminescent substrate that reacts with the enzyme to emit light.
Image the membrane.
A lower signal in CaeB-expressing cells compared to control cells suggests CaeB inhibits apoptosis downstream of the pro-apoptotic protein.
Blot the proteins onto PVDF membranes at a constant voltage of 16 volts for 60 minutes using a trans blot SD semi-dry transfer cell. Then, block the membranes in 10 milliliters of MPT for one hour at room temperature.
Dilute seven microliters of anti-cleaved PARP antibody in seven milliliters of MPT. Incubate the PVDF membrane with the antibody solution overnight at four degrees Celsius. Remove the antibody solution and perform three 10-minute washes with PBS 0.05% Tween 20. Incubate the membranes with 1.4 microliters of horse radish peroxidase conjugated secondary antibodies diluted in seven milliliters of MPT for one hour at room temperature.
After the incubation, wash the membranes three times with PBS 0.05% Tween 20. Detect horseradish peroxidase activity with a commercial kit according to the manufacturer's protocol and apply standard equipment for film development to visualize the protein bands.