WB-Validated AKT1 Lentiviral shRNA Knockdown Kit #V69471

AKT1; AKT Serine/Threonine Kinase 1; RAC; PKB; PRKBA; AKT; V-Akt Murine Thymoma Viral Oncogene Homolog 1; RAC-Alpha Serine/Threonine-Protein Kinase; Protein Kinase B Alpha; Proto-Oncogene C-Akt; Protein Kinase B; RAC-PK-Alpha; EC 2.7.11.1; RAC-ALPHA; PKB Alpha; V-Akt Murine Thymoma Viral Oncogene-Like Protein 1; Serine-Threonine Protein Kinase; Rac Protein Kinase Alpha; RAC-Alpha; PKB-ALPHA; EC 2.7.11; AKT1m

NCBI Gene Entry: 207

1. WB-validated AKT1 shRNA lentiviral particles (1 mL, 1×10⁸TU/mL)
2. Non-targeting shRNA lentiviral particles (1 mL, 1×10⁸TU/mL)
3. Verification Tool: KD-Validated Anti-AKT1 Rabbit mAb #69471 (5 μL)

HeLa

RT-qPCR

Western Blotting (WB)

Lentiviral Particles: Shipped with dry ice. Immediately store the product in a standard freezer at -80°C upon receipt.
Antibody: Shipped with ice packs. Immediately store the product at -20°C upon receipt.

Lentiviral Particles: Store at -80 °C for 1 year.
Antibody: Store at -20 °C for 1 year.

The following protocol uses HeLa cell as an example assuming your cell culture medium is DMEM-based. 

1. Release 0.5 million HeLa 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.
2. 24 h after cell release, pre-warm the shRNA lentiviral medium to 37°C.
3. Discard 1 mL of the original growth medium of the 35 mm dish.
4. Using a serological pipette, gently mix the lentiviral solution 3 times.
5. Carefully add 1 mL of the lentiviral solution to the well. Tip: To prevent splashing, add the solution to the dish along the wall.
6. Add a polybrene stock solution to the culture medium at a final concentRation of 5 μg/mL. Gently swirl the dish to mix.
7. 48 h after cell release, without discarding the original medium, add another 1 mL of lentiviral medium directly into the dish.
8. 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.
9. 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.
10. 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 HeLa cells as a negative control.
11. Allow puromycin selection for 48 h. Almost all wild-type HeLa 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.

This product is for research use only.