Knockdown and knockout are two methods used to silence the target gene of interest. Good implementation of these techniques can enable researchers to better identify and validate meaningful targets in disease treatment. Western blot can be used to confirm the effect of gene knockdown/knockout.
Creative Biolabs is able to effectively and successfully validate knockout/knockdown using the western blot method. As a leader in biotechnology, we offer advanced protocols for western blot validation of target knockout/knockdown to facilitate researchers' research progress in disease research, therapeutic development with innovative products and services.
|Stages||Solutions and Reagents|
|Sample Preparation||Lysis buffer, sample buffer|
|Electrophoresis and Blotting||Loading buffer, running buffer, transfer buffer|
|Immunodetection||Blocking buffer, antibody, antibody buffer, developing solution, stop solution, wash buffer|
Seed cells into 6-well plates for culture. Transfect the cells with a guide vector to achieve knockout/knockdown of the target gene. Collect cells after transfection. Prepare lysates from untargeted control cells and knockout/knockdown cells. Determine the amount of loaded protein and add an equal volume of sample buffer.
Load protein samples onto the gel and run electrophoresis according to the manufacturer's protocol. Transfer proteins from the gel to the PVDF membrane by semi-dry transfer using a transfer system according to the manufacturer's protocol.
Place the membrane in the blotting vessel, add enough blocking solution to cover the entire membrane surface and shake to incubate. This can help to reduce background noise while enhancing the target protein signal. Incubate the membrane with the diluted primary antibody, wash the membrane at the end of the incubation to remove excess primary antibody, continue incubation with a good selection of secondary antibodies in the blocking buffer, finally wash to remove excess secondary antibody.
Select signal imaging reagents for detection. The protein blot lanes in control and knockout/knockdown samples are shown together and show the relative residual intensity after silencing. Downregulation of antibody signaling confirms knockdown/knockout of the corresponding gene.
No bands for both control and validation
Faint bands for both control and validation
Patchy and uneven spots on the blot
Generate bands at unexpected locations
High background on the blot
We hope you will use our recommended protocols and troubleshooting guides to get the best results in knockout/knockdown target confirmation by western blot. To learn about our antibody products and services, contact us to get information on how to get validation success in western blotting.
For research use only. Not intended for any clinical use.