TranslationBlocker Human ErbB4 / Her4 siRNA – DISCONTINUED

DISCONTINUED – Human ErbB4 / Her4 TranslationBlocker™ siRNA Duplex Product Attributes

ErbB4 / Her4 Knockdown Protocol:

1. 1. Starting with cultured cells in growth media seed 12 well plates with 5×10⁴ cells and incubate at 37°C.
   2. Prepare transfection reagent in serum-free growth medium such as Opti-MEM-I. Allow to come to room temperature. Oligofectamine was used to generate the above data in accordance with manufacturer’s instructions.
   3. Dilute siRNA to working concentration in serum-free growth media. For Oligofectamine or similar transfections into 12 well plates, we recommend starting siRNA concentration optimization at around 100-150 pmol siRNA duplex in 200 ul of serum free media.Optimal dilution should be experimentally determined.
   4. Combine diluted siRNA and transfection reagent. Equivalent volumes of prepared Oligofectamine or other transfection reagent and diluted siRNA should be combined and incubated at room temperature for 30 minutes with gentle mixing (optional).
   5. Add prepared siRNA solution to cells. For 12 well plates this will entail adding the solution to cells in approximately 1.1 ml serum-free medium. Additionally, FITC labeled control siRNA (Product Number QC-1F) or positive control siRNA (Beta-Actin; Product Number QC-2) may be used to assess transfection efficiency.
   6. Incubate for several hours in serum free media. The above data was generated with an initial 4 hour incubation in serum-free media.
   7. Add serum containing media to culture without a media change. Generally 1-2 fold of the original volume of siRNA containing serum-free media is added to restore nutrient levels in media. Alternatively, the media may be directly supplemented with serum such as 10% FBS.
   8. Incubate and assay several hours after transfection. Optimal knockdown time should also be experimentally determined. Optionally, a second transfection can be performed 24 hours after the first transfection to enhance knockdown. ErbB4 / Her4 knockdown at the protein level was measured at 72 hours post transfection via western blot.