WB-Validated PFKFB3 Knockdown Cell Lysate Kit

Kit Components:
1. WB-Validated PFKFB3 Knockdown Cell Lysate (100 μg )
2. WT Cell Lysate (100 μg )
3. Verification Tool: KD-Validated Anti-PFKFB3 Rabbit mAb (5 μL)

Manufacturing Process:
The following protocol was used to generate mRNA knockdown cell lysate:
• Release 0.5 million HT-1080 cells into a 35 mm tissue culture dish in 2 mL of the growth medium (DMEM containing 10% FBS and 1% pen/strep). Cell density should reach 50-60% confluence the following day.
• 24 h after cell release, pre-warm the shRNA lentiviral medium to 37°C.
• Discard 1 mL of the original growth medium of the 35 mm dish.
• Using a serological pipette, gently mix the lentiviral solution 3 times.
• Carefully add 1 mL of the lentiviral solution to the well. Tip: To prevent splashing, add the solution to the dish along the wall.
• Add a polybrene stock solution to the culture medium at a final concentration of 5 μg/mL. Gently swirl the dish to mix.
• 48 h after cell release, without discarding the original medium, add another 1 mL of lentiviral medium directly into the dish.
• Add an additional polybrene stock solution into the dish to obtain a final concentration of 5 μg/mL. Tip: Now, the medium in the dish should be a total of 3 mL.
• 72 h after cell release, cells may reach confluence. Trypsinize the cells off the 35 mm dish and culture those cells in a 60 mm dish.
• Add puromycin to the dish at a final concentration of 4 μg/mL. Tip: To assess the efficacy of puromycin selection, culture a dish of wild-type HT-1080 cells as a negative control.
• Allow puromycin selection for 48 h. Almost all wild-type HT-1080 cells should die, while the dish infected with lentiviruses should have some remaining cells.
• Replace the medium with regular growth medium without puromycin and allow the cells to grow to confluence before harvesting or staining.
• Cells were lysed with IntactProtein™ cell/tissue lysis kit and stored in -20°C.

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