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This procedure works but likely requires further optimization for best results.

Contents

Overview

An assay to check for protein-DNA binding.

Materials

Protein-DNA binding

Electrophoresis

Staining

Procedure

Protein-DNA binding

See Knight:Protein DNA binding.

Add 2μL gel loading solution to each 10μL sample.

Electrophoresis

  1. Wear nitrile gloves.
  2. Prepare 1000mL of 0.5X TBE running buffer from 5X stock solution.
  3. Remove the NuPAGE gel from the pouch.
  4. Rinse the gel cassette with deionized water.
  5. Peel the tape from the bottom of the cassette.
  6. Gently pull the comb from the cassette in one smooth motion.
    • If you don't do it smoothly, you can rip the wells.
  7. Rinse the sample wells with 0.5X TBE running buffer.
    • Use a pipetman and pipet to squirt in running buffer.
  8. Invert and shake to remove buffer.
  9. Repeat rinse two more times.
  10. Orient the two gels in the Mini-Cell such that the notched "well" side of the cassette faces inward towards the buffer core.
  11. Seat the gels on the bottom of the Mini-Cell and lock into place with the gel tension wedge.
    • Use the plastic buffer dam if you are only running one gel.
  12. Fill the upper buffer chamber with a small amount of 0.5X TBE running buffer to check tightness of seal.
    • If there is a leak, discard buffer, reseal chamber and try again.
  13. Fill upper buffer chamber. Buffer level should exceed level of the wells. Requires about 200mL
  14. Load 3μL 2-log DNA ladder.
  15. Load samples.
  16. Fill lower buffer chamber at the gap near locking mechanism with 600mL 0.5X TBE running buffer.
    • Should the buffer be chilled?
  17. Run at 100V for 90 minutes.
    • Gel showed some bowing at 100V when run for 65 mins. Drop the voltage?
    • When the Orange G dye front reaches the bottom, the 100bp DNA band is just over halfway down the gel.
  18. Shut off the power.

Staining the gel

  1. Before opening, warm the SYBR green EMSA gel stain concentrate to room temperature.
  2. Vortex and centrifuge tube.
  3. Dilute 5μL of 10,000X SYBR green EMSA gel stain concentrate into 50 mL 0.5X TBE buffer and pour into gel staining tray.
    • Exact amount depends on size of gel staining tray.
  4. Disconnect electrodes.
  5. Remove gels.
  6. Insert a knife in between the two plates and pry the plates apart.
    • You should hear a cracking noise as you break the bond between the two plates.
  7. Gently separate the two plates attempting to leave the gel on the bottom slotted plate.
  8. Cut to separate gel from bottom lip.
  9. Flip over and transfer gel to clean staining tray.
    • Use the lid of a 1000μL pipette tip box.
  10. Incubate ~20 mins on orbital shaker set at 50 rpm, protected from light.
    • Don't use a glass container (glass adsorbs the dye).
    • Don't reuse staining solution.
    • Staining time may vary with gel.
    • Store unused staining solution for 7 days in plastic container at 4°C
  11. Wash the gel in 150mL dH2O for ~10 secs.
  12. Repeat the wash step again.
  13. Wipe transilluminator with soft cloth and dH2O.
  14. Take a gel picture using 300nm tranilluminator. Set the filter wheel to SYBR green.
    • Use a piece of foil to help transfer the gel from the UV box back into the staining tray.
  15. When doing the protocol for the first time ...
    1. Pour ~100mL of the SYPRO Ruby EMSA protein gel stain into the bottle of TCA.
    2. Wait ~5mins for the TCA to dissolve.
    3. Pour the TCA solution back into the bottle containing the rest of the SYPRO Ruby EMSA protein gel stain.
    4. Replace the cap securely.
    5. Mix by inverting at least 10 times.
    6. Check the box on the bottle indicating TCA
    7. Store at room temperature protected from light.
  16. Place the gel in a clean staining tray.
  17. Add 100mL SYPRO Ruby EMSA protein gel stain with TCA.
    • Exact amount depends on size of gel staining tray.
  18. Incubate ~3 hours on orbital shaker set at 50 rpm, protected from light.
    • Don't use a glass container (glass adsorbs the dye). The lid of a 1000μL pipette tip box seems to be about the right size and work well.
    • You can leave the gel in stain overnight.
    • Do not dilute the stain.
    • Do not reuse the staining solution.
  19. Wash the gel in 150mL dH2O for ~10 secs.
  20. Repeat the wash step again.
  21. Destain the gel in 10% methanol, 7% acetic acid for 60 mins.
    • A gel stained overnight may need longer destaining.
  22. Wash the gel in 150mL dH2O for ~10 secs.
  23. Repeat the wash step again.
  24. Wipe transilluminator with soft cloth and dH2O.
  25. Take a gel picture using 300nm tranilluminator. Set the filter wheel to SYPRO Red.
  26. False color and superimpose images.

References

Error fetching PMID 15300760:
Error fetching PMID 12872218:
  1. EMSA kit from Invitrogen

    [Invitrogen]

  2. Error fetching PMID 15300760: [Jing-Electrophoresis-2004]
  3. Error fetching PMID 12872218: [Jing-Proteomics-2003]
All Medline abstracts: PubMed HubMed

Notes

Safety

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