Activated MPK-1 Staining (MAPK-YT, Diphospho-MPK-1)

Prepared by Sudhir Nayak

The following modification to the Schedl Lab dissection and staining protocol applies to Sigma monoclonal M8159 for detecting activated MAP kinase.

Staining for activated MPK-1 with Sigma monoclonal anti-activated MAP kinase (diphosphorylated ERK-1&2) antibody (M8159) in C. elegans is particularly sensitive to dissection time. Dissection times after the addition of levamisole must be limited to 5 min or less. Dissection times of longer than 5 min will result in reduced signal.

After dissection follow the formaldehyde fixation and staining protocol. Use the Sigma monoclonal anti-activated MAP kinase antibody from 1:100 to 1:400 depending on the sensitivity of your detecting equipment. Formaldehyde/methanol (mixed fixation) protocol can also be used if required but nuclear morphology is not as crisp and anti-activated MAP kinase signal is lower than with formaldehyde alone.