Hello,
for my research I would like to establish the Western Blot analysis using C. elegans in our laboratory. More specifically, I am looking for a suitable antibody for UNC-60 the ortholog for Cofilin in C. elegans.
Best regards!
Hi! I do that routinely but not with UNC-60! If you look at Ehud Cohen or Richard Morimoto protocols in their papers they have stable and promising methods for western blotting with worms. One major factor is starting with enough worm number as well as using a suitable protocol. I mainly did NATIVE blots so a self made lysis buffer was used. However you can use the normal RIPA (along with some protease inhibitor) to kick start your experiment. I find alpha tubulin a good loading control in comparison to actin. Depending on the abundance of your protein you can use either the Turbo methods for transferring or use a traditional method. If there are fly antibodies against homolog or ortholog of UNC-60 then use them and if not I would just be brave and order mammalian ones and check out whether there is any signal.
I hope that helps?
Good luck!
I have taken a quick look at major antibody companies but I couldn't find UNC-60 antibody or at least a commercial version of it. However, this group raised antibodies for this protein
Article UNC-60B, an ADF/Cofilin Family Protein, Is Required for Prop...
You might want to drop them an email, they might send you an aliquote of the antibody.
Best of luck
Hi Fabian,
Here is a link to recent protocol specifically for Westerns in elegans from Seung-Jae Lee's lab - https://link.springer.com/protocol/10.1007%2F978-1-4939-7665-2_19
And as for antibodies, wormbase has a couple listed. Links below..
https://wormbase.org/species/all/antibody/[cgc3529]:unc-60_a#01--10
https://wormbase.org/species/all/antibody/[cgc3529]:unc-60_b#01--10
Hope this helps!
Soudabeh Imanikia Waleed Albihlal Francis Amrit
Thank you for your help! That's very nice of you! This information will help me a lot!
Best regards!
Fabian
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