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. 
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 
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the Neuromab antibody never failed me for western blots and IHC. My lysis buffer is: 20 mM tris ph7.4, 50 mM NaCl, 2 mM MgCl2, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS with Protease/phosphotase inhibitors and benzonase. I put the tissues in this buffer and then disrupt with one or two rounds of sonication (no more than 10s because it will heat your sample). then i clarify the lysates by centrifugation (15000xg, 4°C, 10min). I load 10-20µg of total protein on the gel. For the transfer, i use TGS, 20% MeOH, 100V, 1 hour with PVDF membranes 0.45 µm pore. then i block in TBST, 5% milk for 30 min-1h and incubate primary an secondary antibodies in TBST, 5% BSA. This protocol worked for all proteins in my hands (from 330 kDa to 20 kDa).
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Thanks @AubinMoutal. I use RIPA from Sigma with Halt Protease/Phosphatase inhibs using a Precellys 24 CK28 bead beater type homogenizer. My assay is Protein Simple Wes. I don’t sonicate usually because the volumes of DRG lysates are so small. I might try your buffer system and see if it improves things for me.
I ran a Cell Signaling Nav1.7 and Neuromab 1.7 supernatant on the Wes on mDRG and mNerve lysates. Got clear bands, but lower MW than expected. Sometimes this happens on a Wes compared to traditional western. What MW do you normally see?
It’s a clear band, and also the CST ab shows too, although fainter. So not totally sure if true Nav1.7 or something non-specific that is shared.
I run a 4-20% gel in a TGS buffer and usually see a band on or slightly above the 250kDa mark from the PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa. For Nav1.7 the band should be blurry because of the multiple glycosylations on the channel.
For Sonication, it is doable. I put the DRGs in 100µl of the lysis buffer and sonicate with the small probe i have. We’re neighbors now, let me know how i can help!
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