Is your siRNA expected to target all isoforms?

Does your antibody recognize all isoforms?

Yoav Lubelsky The siRNA designed for one isoform. I see the change in functionality due to siRNA treatment. However, I don't see any knockdown in the western blot. Now the question, whether my antibody is probing is the right isoform. Because I don't see any change in intensity compared to control. I used polyclonal and monoclonal both, but have the same results. Now, I am a bit worried if the isoform is knocked down and I am not able to probe it on western blot. So could be the next strategies if you could suggest, it will be a great help.

Thank you in advance

Prem

Can you tell which isoform you detect in the western blot based on the size of the band or are all isoform of similar size?

The antibody datasheet may also include the information of what part of the protein was used to generate the antibody.

By looking at the size I can't tell which isoform it has been detecting, because they all are of same molecular wt. range. I woould carefully look which part of protein is detected by the antibody. But Can I use multiple siRNAs of different isoform in the same reaction mixture to knockdown most of the isoform in such case, because I have seen people have done so in the the research papers.

Any input on it ?

Thanks

You can definitely use multiple siRNAs. We use a pool of 4 as default regardless of isoforms.

Yoav Lubelsky It make sense to me and its very helpful now. Thank you for your valuable input I will do it as per your suggestions. Could you please give an idea how you design the siRNA for a target protein. I am new to this field and I recieved the current siRNA from one my collaborators. Any server, or website or detailed protocol to design siRNA ? please let me know.

Thank you again

Best

Prem

There are several online design tools available.

We generally order SMARTpools from horizon

https://horizondiscovery.com/en/gene-modulation/knockdown/sirna/products/on-targetplus-sirna-reagents