how to see the fluorescence in the daf-16::GFP worms

Model Organism: Worms Methods & Reagents
1

hello
I wana see the daf-16 relocation using the daf-16::GFP worms.In my experiment ,we find the transcription factor daf-16
relocated very quickly even ten miutes in the 2%agar-pad. Can anyone can give me some methoths or some sugestions.
Thanks

2

it’s hot here…ok not as hot as in south east California…but hot enough for me to wonder if my brain is fried and that’s why I can’t make much sense of your question…

So,

  1. you want to use daf-16::gfp worms to monitor the rapid kinetics of DAF-16 ‘relocation’??
    (my most immediate thought is how long the gfp protein ‘tag’ takes to mature…longer than 10 minutes…or is this a express it and move it kind of expt?..and previous work has used a much longer time period probably because of this)

  2. you have conducted experiments already and observed DAF-16 ‘relocation’ after 10 minutes on an agarose pad??
    ( so you have the answer and the means of assaying nuclear relocalisation of DAF-16 already?)

Perhaps you could have another go at your question and describe more completely what you have done and hope to do…without pulling the carpet from under your PI’s feet.?

Steve

3

There’s mountains of literature out there that use daf-16::gfp and look at nuclear ‘translocation’. Are you trying to look at translocation after exposure to a stress or something?

4

I’m guessing that the problem is that DAF-16::GFP is translocating in the control on the agarose pads.

This is surprising, I’ve handled this strain several times and do not see that. Perhaps there’s something about your agarose mixture, anesthetic or the temperature in your microscope room?

Vic

5

I wana see if XX protein toxin make the Transcription factor daf-16 relocate using the TJ356(daf-16::GFP) worms .In my experiment ,we find the transcription factor daf-16
relocated very quickly even ten miutes in the 2%agar-pad. Can anyone can give me some methoths or some sugestions.
Thanks
our protocol:
1 Add E.coli BL21 containing pQE30- XX protein toxin in ENG-IC （plus IPTG and Amp）plate ,Incubate plates overnight at 25℃. E.coli BL21 containing pQE30 in ENG-IC plate as negetive contol.
2 . add 10–20 synchronized L4 worms per plate and incubate at 20℃.
3. Some paper have confirmed the Daf-16 could relocation form Cytoplasm to Nucleus at 30℃ in 15 mins . As a postive control ,we feed the worms in E.coli BL21 containing pQE30 in ENG-IC at 30℃.
5.But the results is confused. The negetive control , worms feed with E.coli BL21 containing pQE30, relocated form Cytoplasm to Nucleus. worms feed with E.coli BL21 containing pQE30- XX protein toxin did not relocate.is the same with the postive control.

6

A few things. Are you sure what you’re seeing is true translocation to the nucleus? If so, how are you sure if your positive control doesn’t work? I’d try heat shocking the worms at 35C for at least 30 minutes for a good positive control.

Are you sure that your protocol is sufficient to induce enough of your recombinant protein? I’ve seen protocols that are much more rigorous:

  1. Grow up O/N cultures at 37C w/ shaking
  2. Take 0.5mL of the culture and add to 4.5 mL LB + Carbenicillin (50ug/mL), grow for an hour at 37C w/ shaking
  3. Add IPTG [50uM final], grow for 3h at 30C w/ shaking
  4. Measure OD600, adjust to a final abs of 2.0
  5. Seed plates and let grow O/N at 25C

That being said, this is more of a general protocol and you will need to optimize your protocol (length of protein induction, concentration of IPTG added to the LB as well as to the ENG-IC plates).

Also, why are you using BL21 (unless it is BL21(DE3))? I think people usually use JM103 or M15 since you need sufficient lac repressor.

7

It’s way too weird that you claimed that your positive control did not behave in its way.

i would go with Snug and suggest if you may put along a heat-shocked worm as a parallel control (to check if your ‘positive control’ is working)
Then you may see the very obvious nuclear localization of DAF-16::GFP.