Knight:Electrophoretic mobility shift assay
In progress! Has not been tested.
An assay to check for protein-DNA binding.
- Binding buffer
- Need to determine composition. See Reshma Shetty/Scratchpad#DNA binding reaction conditions for notes.
- 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
- 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.
- Trichloroacetic acid (TCA)
Incubate protein-DNA mix in binding buffer for 1hr at room temperature???
See Reshma Shetty/Scratchpad#DNA binding reaction conditions for notes.
Add loading solution to sample.
- Wear nitrile gloves.
- Remove the NuPAGE gel from the pouch.
- Rinse the gel cassette with deionized water.
- Peel the tape from the bottom of the cassette.
- Gently pull the comb from the cassette in one smooth motion.
- Rinse the sample wells with 0.5X TBE running buffer.
- Use a pipetman and pipet to squirt in running buffer.
- Invert and shake to remove buffer.
- Repeat rinse two more times.
- Orient the two gels in the Mini-Cell such that the notched "well" side of the cassette faces inward towards the buffer core.
- 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.
- Fill the upper buffer chamber with a small amount of upper buffer chamber running buffer (with antioxidant) to check tightness of seal.
- If there is a leak, discard buffer, reseal chamber and try again.
- Fill upper buffer chamber. Buffer level should exceed level of the wells. Requires about 200mL
- Load samples.
- Load protein molecular weight marker (20 μL per lane).
- Fill lower buffer chamber at the gap near locking mechanism with 600mL 0.5X TBE running buffer.
- Should the buffer be chilled?
- Run at 100V for 90 minutes.
- Shut off the power.
Staining the gel
- Before opening, warm the SYBR green EMSA gel stain concentrate to room temperature.
- Vortex and centrifuge tube.
- Dilute 10μL of 10,000X SYBR green EMSA gel stain concentrate into 100 mL 0.5X TBE buffer and pour into gel staining tray.
- Disconnect electrodes.
- Remove gels.
- 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.
- Gently separate the two plates attempting to leave the gel on the bottom slotted plate.
- Cut to separate gel from bottom lip.
- Flip over and transfer gel to staining tray.
- Incubate ~20 mins on orbital shaker set at 50 rpm for ~20 mins, 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
More to come.
- EMSA kit from Invitrogen
- Jing D, Beechem JM, and Patton WF. . pmid:15300760.
- Jing D, Agnew J, Patton WF, Hendrickson J, and Beechem JM. . pmid:12872218.