WB-Validated SMAD5 Knockdown Cell Lysate Kit#L61516

SMAD Family Member 5; JV5-1; MADH5; Mothers Against Decapentaplegic Homolog 5; DWFC; MAD, Mothers Against Decapentaplegic Homolog 5 (Drosophila); Mothers Against Decapentaplegic, Drosophila, Homolog Of, 5; SMAD, Mothers Against DPP Homolog 5 (Drosophila); MAD, Mothers Against Decapentaplegic Homolog 5; SMAD, Mothers Against DPP Homolog 5; SMA- And MAD-Related Protein 5; Mothers Against DPP Homolog 5; MAD Homolog 5; SMAD 5; HSmad5; Smad5; Dwfc

NCBI Gene Entry:4090

HeLa

RT-qPCR

Western blotting (WB)

Cell Lysate: Shipped with ice packs. 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.

Cell Lysate: Store at -80°C for 1 year.
Antibody: Store at -20 °C for 1 year.

This knockdown cell lysate should be paired with wild-type HeLa cell lysate for use. For Western blotting, we recommend running wild-type and knockdown lysates on the same gel, and loading each well with equal volume and equal amount of total proteins.

Tips: For reducing SDS-PAGE, a final concentration of 2–5% β-mercaptoethanol or 50 mM DTT, plus 0.1% bromophenol blue, must be added to the lysates. Samples should be heated at 95°C for 5 min before loading.

The following protocol was used to generate mRNA knockdown cell lysate:

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.
13. Cells were lysed with IntactProtein™ cell/tissue lysis kit (Cat#415) and stored in -20℃.

1. WB-validated SMAD5 Knockdown Cell Lysate (100 μg )
2. WT Cell Lysate (100 μg )
3. Verification Tool: KD-Validated Anti-SMAD5 Rabbit mAb #61516 (5 μL)

This product is for research use only.