Knight:Electrophoretic mobility shift assay

In progress! Has not been tested.

Overview

An assay to check for protein-DNA binding.

Materials

Protein-DNA binding

• See Knight:Protein DNA binding

Electrophoresis

• Loading solution
  • Comes in EMSA kit if we go that route (light sensitive, stored at -20°C)
  • Alternatively, Tom thinks we could get away with using our typical gel loading buffer.
  • Is this compatible with the DNA retardation gels gels?
• Novex DNA Retardation Gels (manual)
• 0.5X TBE running buffer
  • 44.5 mM Tris base
  • 44.5 mM Boric acid
  • 1 mM EDTA (free acid)
  • pH ? (do not use acid or base to adjust the pH of the solution)
• DNA ladder
  • Use standard 12μL of 2-log ladder with orange G dye? (Seems to bleed a bit into acrylamide). Drop the loading volume down to 6 μL. Then the ladder will give an intensity more comparable to the probe.
• Protein molecular weight standard
• Don't think that people typically run protein molecular weight standards on these gels. (Probably cause you typically only visualize DNA.)
  • Need an unstained protein molecular weight standard (prestained standards interfere with fluorescence of SYPRO Red). Trying Mark12 Unstained Standard. But it might be in the wrong loading buffer. Came out somewhat blotchy. Trying just BSA to check protein staining.

Staining

• SYBR Green EMSA nucleic acid gel stain
  • 10,000X concentrate in dimethylsulfoxide
  • Light sensitive
  • Stored at -20°C
  • Supposedly different from "normal" SYBR Green nucleic acid stain but not sure this is true.
• SYPRO Ruby EMSA protein gel stain
  • Light sensitive.
  • Store at room temperature.
  • Supposedly different from "normal" SYPRO Ruby protein stain but not sure this is true.
  • When mixed with TCA, it is stable for 6 months.
• Trichloroacetic acid (TCA)

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 6μL 2-log DNA ladder.
15. Load samples.
16. Load 5μL Mark12 unstained protein molecular weight standard.
    • Dropped this to 1μL to see if it looks any better.
17. Fill lower buffer chamber at the gap near locking mechanism with 600mL 0.5X TBE running buffer.
    • Should the buffer be chilled?
18. 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.
19. 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 10μL of 10,000X SYBR green EMSA gel stain concentrate into 100 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.
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).
    • 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

1. EMSA kit from Invitrogen

2. Jing D, Beechem JM, and Patton WF. The utility of a two-color fluorescence electrophoretic mobility shift assay procedure for the analysis of DNA replication complexes. Electrophoresis. 2004 Aug;25(15):2439-46. DOI:10.1002/elps.200405994 |
3. Jing D, Agnew J, Patton WF, Hendrickson J, and Beechem JM. A sensitive two-color electrophoretic mobility shift assay for detecting both nucleic acids and protein in gels. Proteomics. 2003 Jul;3(7):1172-80. DOI:10.1002/pmic.200300438 |

Notes

• This kit is not sensitive enough for a complex mixture of protein and RNA. This kit has most optimum results when used with a more purified sample. The analysis of a complex solution with low concentrations of the actual target molecule requires more sensitivity than fluorescence can provide. From Molecular Probes technical assistance.
• Promega has a useful FAQ.

Safety

• Use nitrile gloves while handling acrylamide gels.
• TCA is highly corrosive and hazardous. Use proper personal equipment protection.
• SYBR green and SYPRO red stains must be disposed of as hazardous waste in separate waste streams. The SYPRO red is mixed with trichloroacetic acid and thus should be marked as ignitable and corrosive and the SYBR green is mixed with DMSO and should be marked toxic. (Kathy Gilbert, EHS)

Contact

• Reshma Shetty