Nav1.7 Western Blot for Mice DRG

Hello,

I was wondering if anyone has been successful in doing a western on mice DRG, and if you have, can you please share your protocol. We have been trying with the alomone, cell signaling antibodies with no luck.

Hi there, the Neuromab anti-Nav1.7 (both purified antibody and TC supernatant formats) have worked well for us in transfections of multiple cell lines. Cell lysates are reduced in LDS buffer with BME or DTT, heated to 70 degrees Celsius for 10 mins, and run on a NuPAGE 3-8% Tris-Acetate gel.

I’m sure I’ve used an Alomone trial antibody too though - would there be special sample prep needed for primary cells?

Hello Nivedita,

We have been successful in doing Western in N2a cells, however, the same antibody didn’t work for mouse DRG. :confused: We have now tried 5 antibodies with no luck.

Have you tried different lysis conditions?

Hi,

No the extraction buffer and method is always the same. We use RIPA buffer with a protease cocktail and we can see a lot of protein. Have you ever worked with membrane proteins in DRG? What would you recommend?

Thanks!

I haven’t extracted from DRG myself, but there is a protocol and recipe for CHAPS lysis buffer here that may help: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793798/

Best of luck, please let us know how it goes :slight_smile:

1 Like

Thanks for sharing your insights @Nivedita

@mamocol Let us know how it goes. Good luck!