My software notes

October 19, 2011

NMR protocol (recipe)–make stretch gel for protein RDC measurement

Filed under: recipe_protocol — kpwu @ 7:01 pm
Tags: , ,

I bought the starter kit (NE-373-B-6/4.2) at NewEraNMR for RDC experiment on Varian NMR (with 700 MHz grade) and I decided to stretch the gel a lot, so 6mm-wide gel chamber and “6mm-4.2mm gel funnel” are included.

The NMR tube seems to be more fragile than Wilmad products I often used, so I also ordered additional 5 tubes (both ends are opened).

Since the special point of the NMR tubes used for this stretch gel is “two-end opened”, one can find glass factory to cut the regular NMR then no need to buy this special tube.

To users who can use both Varian and Bruker NMR machines:  you just need to buy some “End Gel Plug” for Bruker machine. It’s not necessary to buy two starter kits.

The picture shown left is an example I made and the bubble near the bottom of gel showed up after 3 days. User may have to to worry the time-dependent changes of the stretched gel.

 

Required solution:

  • 30% acrylamide (w/v) in distilled H2O
  • 0.8% bis-acrylamide (w/v) in distilled H2O
  • 10% APS (w/v), APS = ammonium persulfate
  • TEMED as catalyst
  • Commercial kit to make stretch gel in NMR tube (page 16 in NewEraNMR’s catalog)
Recipe for 1ml mixture: (ul used at here means micro-liter)
  • 6% gel:
    • 30% acrylamide: 200 ul
    • 1% bis-acrylamide: 80 ul
    • 10% APS: 10 ul
    • TEMED: 0.1 ul
    • H2O: 710
  • 4.2 % gel:
    • 30% acrylamide: 140 ul
    • 1% bis-acrylamide: 56 ul
    • 10% APS: 8 ul
    • TEMED: 0.1 ul
    • H2O: 796
You may note that I made the ratios of acrylamide:bis-acrylamide:APS constant between 6% and 4.2% gel. Just try to avoid too much “unreacted” APS and acrylamide inside the gel.
Protocol of gel preparation:
  1. Mix the above recipe and load 800 ul into the gel chamber, wait for 2 hour for gel formation. The “un-used” 200 ul mixture is used to check if the gel is formed.
  2. Gently remove the gel from chamber using the extrude rod provided in the starter kit and use cleaned knife to cut the gel to a proper length. I made both 1.5 and 2mm gels to next step.
  3. Wash the short gels with 15 ml distilled water for 1 hour and repeat 3-5 times. This step is to try to remove unreacted acrylamide, TEMED, and APS inside the gel.
  4. Wash the gel with 15 ml NMR experimental buffer for 1 hour, repeat 3 times. (or do more volume/times and skip step 3)
  5. Soak the gel with 600 ul NMR buffer which has 10% D2O inside and leave it overnight.
  6. Remove the buffer and  transfer~300 ul concentrated NMR sample (10% D2O included) to the tube containing short gel.Wait for free diffusion for overnight.
    I usually use ~1 mM NMR sample and the final protein concentration left in the solution is less than 600 uM suggesting 300-400 uM protein are inside the gel.
  7. Assemble the gel chamber with funnel, load the short gel (with protein and 10% D2O) into it. Follow the guide in the NewEra’s catalogue, it should be easy to move the gel from chamber to NMR tube.
  8. Once the gel is fully inserted into the NMR tube, it’s stretched and quickly insert the “End Gel Plug” then insert the assembled “rod-end up plug” to keep the gel at proper position.

As my personal experiences, 6% gel and 4.2% gel returned me 2H splitting constants of 6.2 Hz and 2.5Hz, respectively.

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