WB-Validated MRPS18B Lentiviral shRNA Knockdown Kit #V65651

MRPS18B; Mitochondrial Ribosomal Protein S18B; C6orf14; MRPS18-2; HSPC183; PTD017; MS40; 28S Ribosomal Protein S18-2, Mitochondrial; 28S Ribosomal Protein S18b, Mitochondrial; Small Ribosomal Subunit Protein BS18b; Small Ribosomal Subunit Protein MS40; MRP-S18-2; MRP-S18-B; Mrps18-B; S18mt-B; Mitochondrial Small Ribosomal Subunit Protein BS18m-B; Mitochondrial Small Ribosomal Subunit Protein BS18b; Mitochondrial Small Ribosomal Subunit Protein MS40; Mitochondrial Ribosomal Protein S18-2; HumanS18a; S18amt

NCBI Gene Entry: 28973

1. WB-Validated MRPS18B shRNA lentiviral particles (4 mL)

2. Non-Target shRNA lentiviral particles (4 mL)

3. Verification Tool: KD-Validated Anti-MRPS18B Rabbit mAb#65651 (5 μL)

HeLa

RT-qPCR

Western Blotting (WB)

Shipped with dry ice. Immediately store the product in a standard freezer at -80°C once received.

Store at -80 °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.
12. Replace the medium with regular growth medium without puromycin and allow the cells to grow to confluence before harvesting or staining.