I’d like to get an antibody made against an ER located C.elegans protein, for western blots, IP IF etc…
Can anyone suggest any companies they have used? or any companies that have experience making anti CE antibodies?
I would prefer monoclonal, but would consider polyclonal
Given the ease of CRISPR/Cas9-mediated genome editing, why not epitope-tag your protein by using an oligonucleotide + Cas9 + sgRNA to insert the necessary codons directly into the gene? Then you can have access to pre-existing high-affinity antibodies for epitopes such as MYC or FLAG.
It may be better than trying to make a new antibody. That involves more steps, takes more time and the mAb you get may not work for all applications.
I’ll give that a go, we are just about to try CRISPR/Cas-9 for knock outs and if that works we will have a go at inserting the epitopes
The Nonet lab developed a monoclonal antibody tool kit for worms, including some to ER proteins (http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0010161). The antibodies are available at the Developmental Studies Hybridoma Bank (http://dshb.biology.uiowa.edu/). We’ve also had success with mammalian antibodies (to Calnexin for example), so those can be worth trying if the conservation is high.
I’ll take a look at those suggestions,
Many mammalian ER-resident proteins have at the C-terminus the ER-retention signal -KDEL or variants. Long time ago I looked for
such proteins in C. elegans and I found that there are more proteins with HDEL than KDEL. You can use the comercially
available anti-KDEL or HDEL antibody. I used the a-HDEL from Santa Cruz in IF and I was not very satisfied, but it might depend on
protocol and tissue.