TranslationBlocker Human ALK tyrosine kinase receptor / CD246 siRNA

Human ALK tyrosine kinase receptor / CD246 TranslationBlocker™ siRNA Duplex Product Attributes

ALK tyrosine kinase receptor / CD246 Knockdown Protocol:

1. 1. Starting with cultured cells in growth media (+10% fetal calf serum (FCS)) seed 12 well plates with 6 x 104 cells and incubate at 37°C for 24 hours.
   2. Prepare transfection reagent in serum-free growth media and allow to come to room temperature. The above data was generated with jetSI in accordance with manufacturer’s instructions. jetSI works well for neural cell populations.
   3. Dilute siRNA to working concentration in serum-free growth media. The above data was generated with 100 pmol of QX1 siRNA duplex in 100 ul of media. Optimal dilution should be experimentally determined.
   4. Combine diluted siRNA and transfection reagent. In this case, 100 ul of jetSI was incubated with 100 ul of diluted siRNA for 30 minutes with gentle mixing (optional).
   5. Add prepared siRNA solution to cells.
   6. Incubate for several hours in serum free media. In the above experiment, a 4 hour incubation was performed.
   7. Add serum containing media to culture. Data above was generated by adding 1x culture volume of media containing 4% FCS (2% final concentration).
   8. Incubate and assay several hour after transfection. An additional transfection may be performed, as above, 24 hours after initial transfection to enhance knockdown. Optimal knockdown time should also be experimentally determined. In the above experimental conditions, ALK knockdown at the protein level was confirmed 84 hours post transfection via western blot.