Knight:Electrophoretic mobility shift assay: Difference between revisions
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**pH ? (do not use acid or base to adjust the pH of the solution) | **pH ? (do not use acid or base to adjust the pH of the solution) | ||
*DNA ladder | *DNA ladder | ||
**Use standard 12μL of 2-log ladder with orange G dye? (Seems to bleed a bit into acrylamide). | **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 | *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.) | *Don't think that people typically run protein molecular weight standards on these gels. (Probably cause you typically only visualize DNA.) | ||
Line 71: | Line 71: | ||
#*''Should the buffer be chilled?'' | #*''Should the buffer be chilled?'' | ||
#Run at 100V for 90 minutes. | #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.'' | |||
#Shut off the power. | #Shut off the power. | ||
Line 94: | Line 96: | ||
#Wipe transilluminator with soft cloth and dH<sub>2</sub>O. | #Wipe transilluminator with soft cloth and dH<sub>2</sub>O. | ||
#Take a gel picture using 300nm tranilluminator. Set the filter wheel to SYBR green. | #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.'' | |||
#When doing the protocol for the first time ... | #When doing the protocol for the first time ... | ||
##Pour ~100mL of the SYPRO Ruby EMSA protein gel stain into the bottle of TCA. | ##Pour ~100mL of the SYPRO Ruby EMSA protein gel stain into the bottle of TCA. |
Revision as of 13:45, 15 September 2006
In progress! Has not been tested.
Overview
An assay to check for protein-DNA binding.
Materials
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.
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
- Wear nitrile gloves.
- Prepare 1000mL of 0.5X TBE running buffer from 5X stock solution.
- 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.
- If you don't do it smoothly, you can rip the wells.
- 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 0.5X TBE running buffer 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 12μL 2-log DNA ladder.
- Load samples.
- Load 5μL Mark12 unstained protein molecular weight standard.
- 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.
- 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.
- 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.
- Exact amount depends on size of 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 clean staining tray.
- 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
- Wash the gel in 150mL dH2O for ~10 secs.
- Repeat the wash step again.
- Wipe transilluminator with soft cloth and dH2O.
- 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.
- When doing the protocol for the first time ...
- Pour ~100mL of the SYPRO Ruby EMSA protein gel stain into the bottle of TCA.
- Wait ~5mins for the TCA to dissolve.
- Pour the TCA solution back into the bottle containing the rest of the SYPRO Ruby EMSA protein gel stain.
- Replace the cap securely.
- Mix by inverting at least 10 times.
- Check the box on the bottle indicating TCA
- Store at room temperature protected from light.
- Place the gel in a clean staining tray.
- Add 100mL SYPRO Ruby EMSA protein gel stain with TCA.
- Exact amount depends on size of gel staining tray.
- 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.
- Wash the gel in 150mL dH2O for ~10 secs.
- Repeat the wash step again.
- Destain the gel in 10% methanol, 7% acetic acid for 60 mins.
- A gel stained overnight may need longer destaining.
- Wash the gel in 150mL dH2O for ~10 secs.
- Repeat the wash step again.
- Wipe transilluminator with soft cloth and dH2O.
- Take a gel picture using 300nm tranilluminator. Set the filter wheel to SYPRO Red.
- False color and superimpose images.
References
-
EMSA kit from Invitrogen
- 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 |
- 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 mixuture 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.
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. (Kathy Gilbert, EHS)