Knight:Electrophoretic mobility shift assay: Difference between revisions
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**Supposedly different from "normal" SYBR Green nucleic acid stain but not sure this is true. | **Supposedly different from "normal" SYBR Green nucleic acid stain but not sure this is true. | ||
*SYPRO Ruby EMSA protein gel stain | *SYPRO Ruby EMSA protein gel stain | ||
**Light sensitive | **Light sensitive. | ||
**Store at room temperature | **Store at room temperature. | ||
**Supposedly different from "normal" SYPRO Ruby protein stain but not sure this is true. | **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) | *Trichloroacetic acid (TCA) | ||
Line 67: | Line 68: | ||
#Vortex and centrifuge tube. | #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. | #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. | #Disconnect electrodes. | ||
#Remove gels. | #Remove gels. | ||
Line 73: | Line 75: | ||
#Gently separate the two plates attempting to leave the gel on the bottom slotted plate. | #Gently separate the two plates attempting to leave the gel on the bottom slotted plate. | ||
#Cut to separate gel from bottom lip. | #Cut to separate gel from bottom lip. | ||
#Flip over and transfer gel to staining tray. | #Flip over and transfer gel to clean staining tray. | ||
#Incubate ~20 mins on orbital shaker set at 50 rpm | #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 use a glass container (glass adsorbs the dye). | ||
#*Don't reuse staining solution. | #*Don't reuse staining solution. | ||
#*Staining time may vary with gel. | #*Staining time may vary with gel. | ||
#*Store unused staining solution for 7 days in plastic container at 4°C | #*Store unused staining solution for 7 days in plastic container at 4°C | ||
#Wash the gel in 150mL dH<sub>2</sub>O for ~10 secs. | |||
#Repeat the wash step again. | |||
#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. | |||
#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 dH<sub>2</sub>O 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 dH<sub>2</sub>O for ~10 secs. | |||
#Repeat the wash step again. | |||
#Wipe transilluminator with soft cloth and dH<sub>2</sub>O. | |||
#Take a gel picture using 300nm tranilluminator. (Figure out what filter to use.) | |||
#False color and superimpose images. | |||
==References== | ==References== | ||
Line 88: | Line 117: | ||
#Jing-Proteomics-2003 pmid=12872218 | #Jing-Proteomics-2003 pmid=12872218 | ||
</biblio> | </biblio> | ||
===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. | |||
==Contact== | ==Contact== | ||
*[[Reshma Shetty]] | *[[Reshma Shetty]] |
Revision as of 12:29, 28 August 2006
In progress! Has not been tested.
Overview
An assay to check for protein-DNA binding.
Materials
Protein-DNA binding
- Binding buffer
- Need to determine composition. See Reshma Shetty/Scratchpad#DNA binding reaction conditions for notes.
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
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
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.
Electrophoresis
- 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.
- 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.
- 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. (Figure out what filter to use.)
- 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.