Knight:Electrophoretic mobility shift assay

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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)
-
*Loading solution
 
-
**Tom thinks we could get away with using our [[Knight:Loading dye|typical gel loading buffer]].
 
-
*Binding buffer
 
-
**Need to determine composition.  See [[Reshma Shetty/Scratchpad#DNA binding reaction conditions]] for notes.
 
==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 13:48, 28 August 2006

In progress! Has not been tested.

Contents

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

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

  1. Wear nitrile gloves.
  2. Remove the NuPAGE gel from the pouch.
  3. Rinse the gel cassette with deionized water.
  4. Peel the tape from the bottom of the cassette.
  5. Gently pull the comb from the cassette in one smooth motion.
  6. Rinse the sample wells with 0.5X TBE running buffer.
    • Use a pipetman and pipet to squirt in running buffer.
  7. Invert and shake to remove buffer.
  8. Repeat rinse two more times.
  9. Orient the two gels in the Mini-Cell such that the notched "well" side of the cassette faces inward towards the buffer core.
  10. 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.
  11. 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.
  12. Fill upper buffer chamber. Buffer level should exceed level of the wells. Requires about 200mL
  13. Load samples.
  14. Load protein molecular weight marker (20 μL per lane).
  15. Fill lower buffer chamber at the gap near locking mechanism with 600mL 0.5X TBE running buffer.
    • Should the buffer be chilled?
  16. Run at 100V for 90 minutes.
  17. 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.
  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 staining tray.
  10. 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

  1. EMSA kit from Invitrogen [Invitrogen]
  2. Jing D, Beechem JM, and Patton WF. . pmid:15300760. PubMed HubMed [Jing-Electrophoresis-2004]
  3. Jing D, Agnew J, Patton WF, Hendrickson J, and Beechem JM. . pmid:12872218. PubMed HubMed [Jing-Proteomics-2003]
All Medline abstracts: PubMed HubMed

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