Knight:Electrophoretic mobility shift assay: Difference between revisions
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==Materials== | ==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 [[Knight:Loading dye|typical gel loading buffer]]. | |||
**Is this compatible with the DNA retardation gels gels? | |||
*[http://www.invitrogen.com/content/sfs/manuals/dnaretardationgel_card.pdf Novex DNA Retardation Gels] ([http://www.invitrogen.com/content/sfs/manuals/electrophoresisguide_man.pdf manual]) | |||
*0.5X TBE running buffer | |||
===Staining=== | |||
*SYBR Green EMSA nucleic acid gel stain | *SYBR Green EMSA nucleic acid gel stain | ||
**10,000X concentrate in dimethylsulfoxide | **10,000X concentrate in dimethylsulfoxide | ||
**Light sensitive | |||
**Stored at -20°C | **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 | *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. | |||
*Trichloroacetic acid (TCA) | *Trichloroacetic acid (TCA) | ||
==Procedure== | ==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. | |||
#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. | |||
==References== | ==References== |
Revision as of 10:48, 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.
- 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.
- 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.
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 |