Knight:Electrophoretic mobility shift assay

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<font color=red>In progress!  Has not been tested.</font>
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<font color=red>This procedure works but likely requires further optimization for best results.</font>
==Overview==
==Overview==
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===Electrophoresis===
===Electrophoresis===
*Loading solution  
*Loading solution  
-
**Comes in EMSA kit if we go that route (light sensitive, stored at -20&deg;C)
+
**Light sensitive
 +
**Stored at -20&deg;C
 +
**Comes in EMSA kit from Invitrogen ([http://probes.invitrogen.com/media/pis/mp33075.pdf manual], catalog number - [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=2538&CMP=LEC-GCMSSEARCH&HQS=EMSA E-33075])
**Alternatively, Tom thinks we could get away with using our [[Knight:Loading dye|typical gel loading buffer]].
**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?
**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])
+
*[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], catalog number - [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=32194&CMP=LEC-GCMSSEARCH&HQS=DNA%20retardation EC6365BOX] (10 well) or [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=32194&CMP=LEC-GCMSSEARCH&HQS=DNA%20retardation EC63652BOX] (12 well))
*0.5X TBE running buffer
*0.5X TBE running buffer
**44.5 mM Tris base
**44.5 mM Tris base
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**1 mM EDTA (free acid)
**1 mM EDTA (free acid)
**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)
 +
**Can make yourself or buy from Invitrogen (catalog number LC6675)
*DNA ladder
*DNA ladder
-
**Use standard 12&mu;L of 2-log ladder with orange G dye?
+
**Use standard 12&mu;L of 2-log ladder with orange G dye? ''Seems to bleed a bit into acrylamideDrop the loading volume down to 2 &mu;LThen the ladder will give an intensity more comparable to the 50ng probe but it still bleeds a bit.''
-
*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===
===Staining===
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**Light sensitive
**Light sensitive
**Stored at -20&deg;C
**Stored at -20&deg;C
 +
**Comes in EMSA kit from Invitrogen ([http://probes.invitrogen.com/media/pis/mp33075.pdf manual], catalog number - [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=2538&CMP=LEC-GCMSSEARCH&HQS=EMSA E-33075])
**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.
 +
**"Normal" SYBR green nucleic acid stain seems to work just as well.
*SYPRO Ruby EMSA protein gel stain
*SYPRO Ruby EMSA protein gel stain
**Light sensitive.
**Light sensitive.
**Store at room temperature.
**Store at room temperature.
 +
**Comes in EMSA kit from Invitrogen ([http://probes.invitrogen.com/media/pis/mp33075.pdf manual], catalog number - [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=2538&CMP=LEC-GCMSSEARCH&HQS=EMSA E-33075])
**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.
**When mixed with TCA, it is stable for 6 months.
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===Electrophoresis===
===Electrophoresis===
#Wear nitrile gloves.
#Wear nitrile gloves.
-
#Prepare 1000mL of 0.5X TBE running buffer from 5X or 10X stock solution.
+
#Prepare 1000mL of 0.5X TBE running buffer from 5X stock solution.
#Remove the NuPAGE gel from the pouch.
#Remove the NuPAGE gel from the pouch.
#Rinse the gel cassette with deionized water.
#Rinse the gel cassette with deionized water.
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#*If there is a leak, discard buffer, reseal chamber and try again.
#*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
#Fill upper buffer chamber.  Buffer level should exceed level of the wells.  Requires about 200mL
-
#Load 12&mu;L 2-log DNA ladder.
+
#Load 3&mu;L 2-log DNA ladder.
#Load samples.
#Load samples.
-
#Load protein molecular weight marker (? &mu;L per lane).
 
#Fill lower buffer chamber at the gap near locking mechanism with 600mL 0.5X TBE running buffer.
#Fill lower buffer chamber at the gap near locking mechanism with 600mL 0.5X TBE running buffer.
#*''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.
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#''Before opening'', warm the SYBR green EMSA gel stain concentrate to room temperature.
#''Before opening'', warm the SYBR green EMSA gel stain concentrate to room temperature.
#Vortex and centrifuge tube.
#Vortex and centrifuge tube.
-
#Dilute 10&mu;L of 10,000X SYBR green EMSA gel stain concentrate into 100 mL 0.5X TBE buffer and pour into gel staining tray.
+
#Dilute 5&mu;L of 10,000X SYBR green EMSA gel stain concentrate into 50 mL 0.5X TBE buffer and pour into gel staining tray.
#*Exact amount depends on size of gel staining tray.   
#*Exact amount depends on size of gel staining tray.   
#Disconnect electrodes.
#Disconnect electrodes.
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#Cut to separate gel from bottom lip.
#Cut to separate gel from bottom lip.
#Flip over and transfer gel to clean staining tray.
#Flip over and transfer gel to clean staining tray.
 +
#*''Use the lid of a 1000&mu;L pipette tip box.''
#Incubate ~20 mins on orbital shaker set at 50 rpm, protected from light.
#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).
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#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.
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#*Exact amount depends on size of gel staining tray.
#*Exact amount depends on size of gel staining tray.
#Incubate ~3 hours on orbital shaker set at 50 rpm, protected from light.
#Incubate ~3 hours 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).  The lid of a 1000&mu;L pipette tip box seems to be about the right size and work well.
#*You can leave the gel in stain overnight.
#*You can leave the gel in stain overnight.
#*Do not dilute the stain.
#*Do not dilute the stain.
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</biblio>
</biblio>
-
===Notes===
+
==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.
+
*''This kit is not sensitive enough for a complex mixture 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.
 +
*Promega has a useful [http://www.promega.com/faq/gelshfaq.html FAQ].
 +
*Don't bother running a protein standard.  It doesn't come out very well (either faint or blotchy depending on amount).
 +
*To date, the best staining tray I've found is the lid of a 1000&mu;L pipette tip box.  Then use a piece of mesh that just fits inside the lide to either keep the gel in place while changing solutions or to move the gel to and from the light box.  This method requires smaller volumes of stain than the staining tray from Invitrogen.
==Safety==
==Safety==
*Use nitrile gloves while handling acrylamide gels.
*Use nitrile gloves while handling acrylamide gels.
*TCA is highly corrosive and hazardous.  Use proper personal equipment protection.
*TCA is highly corrosive and hazardous.  Use proper personal equipment protection.
 +
*''SYBR green and SYPRO red stains must be disposed of as hazardous waste in separate waste streams.  The SYPRO red is mixed with trichloroacetic acid and thus should be marked as ignitable and corrosive and the SYBR green is mixed with DMSO and should be marked toxic.''  (Kathy Gilbert, EHS)
==Contact==
==Contact==
*[[Reshma Shetty]]
*[[Reshma Shetty]]
 +
 +
[[Category:Protocol]]
 +
[[Category:In vitro]]
 +
[[Category:Protein]]
 +
[[Category:DNA]]

Current revision

This procedure works but likely requires further optimization for best results.

Contents

Overview

An assay to check for protein-DNA binding.

Materials

Protein-DNA binding

Electrophoresis

  • Loading solution
    • Light sensitive
    • Stored at -20°C
    • Comes in EMSA kit from Invitrogen (manual, catalog number - E-33075)
    • 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, catalog number - EC6365BOX (10 well) or EC63652BOX (12 well))
  • 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)
    • Can make yourself or buy from Invitrogen (catalog number LC6675)
  • 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 2 μL. Then the ladder will give an intensity more comparable to the 50ng probe but it still bleeds a bit.

Staining

  • SYBR Green EMSA nucleic acid gel stain
    • 10,000X concentrate in dimethylsulfoxide
    • Light sensitive
    • Stored at -20°C
    • Comes in EMSA kit from Invitrogen (manual, catalog number - E-33075)
    • Supposedly different from "normal" SYBR Green nucleic acid stain but not sure this is true.
    • "Normal" SYBR green nucleic acid stain seems to work just as well.
  • SYPRO Ruby EMSA protein gel stain
    • Light sensitive.
    • Store at room temperature.
    • Comes in EMSA kit from Invitrogen (manual, catalog number - E-33075)
    • 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

  1. Wear nitrile gloves.
  2. Prepare 1000mL of 0.5X TBE running buffer from 5X stock solution.
  3. Remove the NuPAGE gel from the pouch.
  4. Rinse the gel cassette with deionized water.
  5. Peel the tape from the bottom of the cassette.
  6. Gently pull the comb from the cassette in one smooth motion.
    • If you don't do it smoothly, you can rip the wells.
  7. Rinse the sample wells with 0.5X TBE running buffer.
    • Use a pipetman and pipet to squirt in running buffer.
  8. Invert and shake to remove buffer.
  9. Repeat rinse two more times.
  10. Orient the two gels in the Mini-Cell such that the notched "well" side of the cassette faces inward towards the buffer core.
  11. 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.
  12. 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.
  13. Fill upper buffer chamber. Buffer level should exceed level of the wells. Requires about 200mL
  14. Load 3μL 2-log DNA ladder.
  15. Load samples.
  16. Fill lower buffer chamber at the gap near locking mechanism with 600mL 0.5X TBE running buffer.
    • Should the buffer be chilled?
  17. 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.
  18. 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 5μL of 10,000X SYBR green EMSA gel stain concentrate into 50 mL 0.5X TBE buffer and pour into gel staining tray.
    • Exact amount depends on size of 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 clean staining tray.
    • Use the lid of a 1000μL pipette tip box.
  10. 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
  11. Wash the gel in 150mL dH2O for ~10 secs.
  12. Repeat the wash step again.
  13. Wipe transilluminator with soft cloth and dH2O.
  14. 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.
  15. When doing the protocol for the first time ...
    1. Pour ~100mL of the SYPRO Ruby EMSA protein gel stain into the bottle of TCA.
    2. Wait ~5mins for the TCA to dissolve.
    3. Pour the TCA solution back into the bottle containing the rest of the SYPRO Ruby EMSA protein gel stain.
    4. Replace the cap securely.
    5. Mix by inverting at least 10 times.
    6. Check the box on the bottle indicating TCA
    7. Store at room temperature protected from light.
  16. Place the gel in a clean staining tray.
  17. Add 100mL SYPRO Ruby EMSA protein gel stain with TCA.
    • Exact amount depends on size of gel staining tray.
  18. Incubate ~3 hours on orbital shaker set at 50 rpm, protected from light.
    • Don't use a glass container (glass adsorbs the dye). The lid of a 1000μL pipette tip box seems to be about the right size and work well.
    • You can leave the gel in stain overnight.
    • Do not dilute the stain.
    • Do not reuse the staining solution.
  19. Wash the gel in 150mL dH2O for ~10 secs.
  20. Repeat the wash step again.
  21. Destain the gel in 10% methanol, 7% acetic acid for 60 mins.
    • A gel stained overnight may need longer destaining.
  22. Wash the gel in 150mL dH2O for ~10 secs.
  23. Repeat the wash step again.
  24. Wipe transilluminator with soft cloth and dH2O.
  25. Take a gel picture using 300nm tranilluminator. Set the filter wheel to SYPRO Red.
  26. False color and superimpose images.

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

Notes

  • This kit is not sensitive enough for a complex mixture 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.
  • Promega has a useful FAQ.
  • Don't bother running a protein standard. It doesn't come out very well (either faint or blotchy depending on amount).
  • To date, the best staining tray I've found is the lid of a 1000μL pipette tip box. Then use a piece of mesh that just fits inside the lide to either keep the gel in place while changing solutions or to move the gel to and from the light box. This method requires smaller volumes of stain than the staining tray from Invitrogen.

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 in separate waste streams. The SYPRO red is mixed with trichloroacetic acid and thus should be marked as ignitable and corrosive and the SYBR green is mixed with DMSO and should be marked toxic. (Kathy Gilbert, EHS)

Contact

Personal tools