Protein Isolation for Western Blots (protocols)?

Experimental Techniques
1

Hey all,
Looking to do some protein work soon. I have some personal protocols from grad school but it’d be nice to see what people are doing.

If you’re willing, share your protocol for:

Mouse

  • Spinal cord
  • DRG
  • Nerve

Human

  • DRG
  • Nerve

I made video a few years back on Mouse DRG lysate prep based on the protocol @tberta taught me

I used to use a BeadBeater as well for many things. I like to avoid the sonicator whenever possible, but I don’t have the bead beater anymore.

@tberta @sshiers @CandlerPaige @thicunha @ShanTan

1 Like
2

Hey all. I’ll answer my own question. I used a Dounce homogenizer for hDRG and hSC, and
https://www.dwk.com/na/kimble-kontes-duall-micro-tissue-grinder-02-ml-885470-0000 for mouse DRG and nerve.

RIPA + complete mini + halt protease inhibitor

Looked at TUBB3 by capillary electrophoresis immunoassay (like a Western).

Looks good! Can’t say that all proteins were extracted optimally but it’s good enough to get some protein.

image
image1163×1000 51.1 KB

I’ll post protocols soon.

2 Likes
3

Greetings,
For mouse DRG - would you pool multiple DRG together to have enough protein to extract? If so, how many? I was hoping that I would be able to extract enough protein from perhaps L4 and L5 DRG from a few animals, but worry about low protein concentration. Also would you mind sharing your protocol?

4

We pooled multiple mouse Lumbar DRGs (6 pairs ) and lysed in 200 uL RIPA+0.5% SDS buffer. We usually obtain 300 ug total protein.

2 Likes