| Product Name |
WB-Validated ITGB3 Lentiviral shRNA Knockdown Kit |
| Aliases |
ITGB3;Integrin Subunit Beta 3; GPIIIa; CD61; GP3A; Integrin, Beta 3 (Platelet Glycoprotein IIIa, Antigen CD61); Platelet Membrane Glycoprotein IIIa; Integrin Beta-3; Antigen CD61; Integrin Beta Chain, Beta 3; Platelet Glycoprotein IIIa; Integrin Beta 3; CD61 Antigen; BDPLT16; BDPLT24; BDPLT2; GT2; GT |
| Background |
Gene Name: ITGB3
NCBI Gene Entry: 3690
|
| Storage |
Store at -80 °C for one year. |
| Kit Components |
1. WB-Validated ITGB3 shRNA lentiviral particles (4 mL) 2. Non-Target shRNA lentiviral particles (4 mL) 3. Verification Tool: KD-Validated Anti-Integrin beta 3 Rabbit mAb #68733 (5 μL) |
| Tested Cell Line |
C2C12 |
| Validation Methods |
RT-qPCR; Western Blotting (WB) |
| Shipping |
Shipped with dry ice. Immediately store the product in a standard freezer at -80°C upon receipt. |
| Instructions For Use |
The following protocol uses C2C12 cell as an example assuming your cell culture medium is DMEM-based.
1.Release 0.5 million C2C12 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 C2C12 cells as a negative control.
11.Allow puromycin selection for 48 h. Almost all wild-type C2C12 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. |
| Note |
1. This product is for research use only.
2. This product is only supplied to end users.
3. Do not use this product for commercial.
4. The shRNA sequence was designed based on the human gene sequence. Since there was no antibody available with species reactivity of Human, the murine C2C12 cell line was chosen to validate the gene knockdown effectiveness using an antibody product with species reactivity of Mouse. |
