Knight:NuPAGE electrophoresis

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==Purpose==
==Purpose==
To run a denaturing protein gel.  Note that a preferable version of this protocol is available at [[Sauer:bis-Tris SDS-PAGE, the very best]] (recommended by Kathleen).  But since we seem to already have the Invitrogen kit contents in lab, this protocol describes use of the Invitrogen system.
To run a denaturing protein gel.  Note that a preferable version of this protocol is available at [[Sauer:bis-Tris SDS-PAGE, the very best]] (recommended by Kathleen).  But since we seem to already have the Invitrogen kit contents in lab, this protocol describes use of the Invitrogen system.
-
 
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Note that the gel and running buffer were chosen for a 13kD protein.
 
==Materials==
==Materials==
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*NuPAGE Novex 10% Bis-Tris Gel 1.0 mm, 10 well
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*NuPAGE Novex 10% Bis-Tris Gel 1.0 mm, 10 well (for small proteins e.g. 13kD) or NuPAGE Novex 4-12% Bis-Tris Gel 1.0 mm, 10 well (for medium proteins e.g. 50-60kD)
*[https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=792& XCell ''SureLock'' Mini-Cell electrophoresis apparatus]
*[https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=792& XCell ''SureLock'' Mini-Cell electrophoresis apparatus]
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*20X MES Running Buffer  
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*20X MES or [[MOPS]] Running Buffer  
*NuPAGE LDS Sample Buffer (4X)
*NuPAGE LDS Sample Buffer (4X)
*NuPAGE Reducing Agent (10X)
*NuPAGE Reducing Agent (10X)
*NuPAGE Antioxidant
*NuPAGE Antioxidant
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*SeeBlue Plus2 Pre-Stained Standard
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*[https://catalog.invitrogen.com/productImages/1900/1896.GIF SeeBlue Plus2 Pre-Stained Standard]
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*Gel drying solution
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*[http://www.invitrogen.com/content.cfm?pageid=9869 Invitrogen SimplyBlue SafeStain]
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**20% ethanol
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**10% glycerol
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*Gel drying frames from Diversified Biotech
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*Cellophane sheets from Diversified Biotech
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==Procedure==
==Procedure==
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#Prepare 1000mL 1X NuPAGE SDS running buffer
#Prepare 1000mL 1X NuPAGE SDS running buffer
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#*50mL 20X MES Running Buffer
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#*50mL 20X MES Running Buffer (for very small proteins e.g. 13kD) or 20X MOPS Running Buffer (for medium-sized proteins e.g. 50-60kD).
#*950mL deionized water
#*950mL deionized water
#Mix well.
#Mix well.
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#*''Use lid of a 1000μL pipette tip box.''
#*''Use lid of a 1000μL pipette tip box.''
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You have two options for staining at this point.
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You have three options for staining at this point.
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====Fast, microwave protocol====
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#[[Knight:NuPAGE electrophoresis/Fast staining]] (fast but less sensitive protocol)
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#Add 100mL deionized water in staining tray.
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#[[Knight:NuPAGE electrophoresis/Slow staining]] (slow but more sensitive protocol)
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#Microwave staining tray loosely covered for 1 min on high.
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#[[Knight:NuPAGE electrophoresis/Hybrid staining]] (allows you to see results immediately but take gel picture when the gel is stained more thoroughly)
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#Shake staining tray for 1 min on orbital shaker at room temperature.
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#Discard water.
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#Repeat steps 1-4 two more times.
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#Add 20mL SimplyBlue SafeStain.
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#*Need 100mL for staining tray.
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#Microwave staining tray loosely covered for 45 secs on high.
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#*1 min seemed a bit too long.
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#Shake staining tray for 5 mins on orbital shaker at room temperature.
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#Discard SimplyBlue SafeStain.
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#Add 100mL deionized water.
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#Add a kimwipe.
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#Shake staining tray for 10 mins on orbital shaker at room temperature.
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#Add 20mL 20% NaCl for at least 5 mins.
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#Gel can be stored in salt solution for several weeks.
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-
This protocol seems to work pretty well.
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See [[Knight:NuPAGE electrophoresis/Gel drying]] for instructions on how to dry and preserve the gel.
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====Slow, room temperature protocol====
 
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#Add 100mL deionized water in staining tray.
 
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#Shake staining tray for 5 min on orbital shaker at room temperature.
 
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#Discard water.
 
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#Repeat steps 1-3 two more times.
 
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#Add 20mL SimplyBlue SafeStain.
 
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#Shake staining tray for 1 hr on orbital shaker at room temperature.
 
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#Add 100mL deionized water.
 
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#Shake staining tray for 1 hr or more on orbital shaker at room temperature.
 
-
This protocol worked less well for me.  However, I learned later that the typical trick people use is to include a kimwipe in the destain step to help soak up the stain. This will likely improve the signal to noise on the gel.
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===Marker Sizes===
 +
* The SeeBlue Plus 2 marker (blue unless another color is indicated); 10 μL samples have around 1-2 μg of protein per band
 +
** Myosin  188 KD (MES buffer) 191 KD (MOPS buffer)
 +
** Phosphorylase B  98 KD (MES buffer) 97 KD (MOPS buffer) (orange)
 +
** BSA 62 KD (MES buffer) 64 KD (MOPS buffer)
 +
** Glutamic dehydrogenase 49 KD (MES buffer) 51 KD (MOPS buffer)
 +
** Alcohol dehydrogenase 38 KD (MES buffer) 39 KD (MOPS buffer)
 +
** Carbonic anhydrase 28 KD
 +
** Myoglobin 17 KD (MES buffer) 19 KD (MOPS buffer) (purple)
 +
** Lysozyme 14 KD
 +
** Aprotinin 9 KD
 +
** Insulin B chain 3 KD
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===Drying the gel===
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* [http://www.invitrogen.com/etc/medialib/en/images/mainbody/punchout.Par.89536.Image.-1.-1.1.gif Marker chart for Invitrogen Bis-Tris and Tris-Acetate Nupage gels]
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#Equilibrate gel in gel drying solution for at least 30 mins.
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#*Reduces gel swelling and results in a more flexible dried gel.
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#Place two cellophane sheets in water for 1-2 mins.
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#*Cellophane may appear cloudy but will clear upon drying.
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#Lay one sheet of cellophane on solid back plate, bevelled edge down. Avoid air bubbles.
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#Place gel on cellophane. Avoid air bubbles.
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#*Air bubbles can cause cracking.
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#Pipet 1-2 mLs of gel drying solution on top of gel.
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#Layer a second wet sheet of cellophane on top of gel.  Match edges with edges of back plate.  Roll cellophane from bottom of gel towards the wells helps avoid air bubbles.
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#Place open frame over stack, bevelled edge up.  Match edges of back plate.  Frame should cover all edges of cellophane.
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#Attach plastic clips to all four sides.
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#Leave assembly to dry overnight horizontally.
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#Remove clips and pry apart assembly.
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#Peel dried gel/cellophane sandwich from back plate.
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#Trim off excess cellophane immediately to avoid curling.
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==Notes==
==Notes==
*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.
*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.
 +
*There are several ways to screw up this protocol, I list them here in the hopes that it may help people avoid these trivial mistakes.
 +
*#Failing to remove the seal at the bottom of the precast gel
 +
*#The inner and outer chambers of running buffer are not sealed from one another.
 +
*#Reverse the electrodes in the power supply.
 +
*#Putting the gel in backwards in the apparatus.
==Safety==
==Safety==
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#NuPAGETechnicalGuide [http://www.invitrogen.com/content/sfs/manuals/nupage_tech_man.pdf NuPAGE Technical Guide]
#NuPAGETechnicalGuide [http://www.invitrogen.com/content/sfs/manuals/nupage_tech_man.pdf NuPAGE Technical Guide]
</biblio>
</biblio>
 +
 +
[[Category:Protein]] [[Category:In vitro]] [[Category:Protocol]]

Current revision

For a much cheaper version of this protocol, see Sauer:bis-Tris SDS-PAGE, the very best.

Contents

Purpose

To run a denaturing protein gel. Note that a preferable version of this protocol is available at Sauer:bis-Tris SDS-PAGE, the very best (recommended by Kathleen). But since we seem to already have the Invitrogen kit contents in lab, this protocol describes use of the Invitrogen system.

Materials

Procedure

Running buffer preparation

Do this step first.

  1. Prepare 1000mL 1X NuPAGE SDS running buffer
    • 50mL 20X MES Running Buffer (for very small proteins e.g. 13kD) or 20X MOPS Running Buffer (for medium-sized proteins e.g. 50-60kD).
    • 950mL deionized water
  2. Mix well.
  3. Set aside 200mL buffer for use in Upper (Inner) buffer chamber).
    • Add 500μL NuPAGE Antioxidant immediately before running the gel.
    • Mix well.

Sample preparation

  1. Wells can accommodate 25μL loading volume.
    • Likely they can accommodate more ... maybe 40μL.
  2. Let sample buffer warm to room temperature.
  3. Each sample should contain
    • 13 μL protein sample (max 0.5μg per band)
    • 5 μL NuPAGE LDS Sample Buffer
    • 2 μL NuPAGE Reducing Agent
  4. Heat samples at 70°C for 10 mins.

Set up the gel apparatus during the 10 mins sample heating step.

Note that prestained molecular weight marker doesn't need any preparation.

Running the gel

  1. Wear 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 1X NuPAGE SDS 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 but 10μL also seems to work).
  15. Fill lower buffer chamber at the gap near locking mechanism with 600mL NuPAGE SDS running buffer.
  16. Run at 200V for 30 minutes.

Staining the gel

  1. Shut off the power.
  2. Disconnect electrodes.
  3. Remove gels.
  4. 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.
  5. Gently separate the two plates attempting to leave the gel on the bottom slotted plate.
  6. Cut to separate gel from bottom lip.
  7. Flip over and transfer gel to staining tray that has been prefilled with 100mL deionized water (see below).
    • Use lid of a 1000μL pipette tip box.

You have three options for staining at this point.

  1. Knight:NuPAGE electrophoresis/Fast staining (fast but less sensitive protocol)
  2. Knight:NuPAGE electrophoresis/Slow staining (slow but more sensitive protocol)
  3. Knight:NuPAGE electrophoresis/Hybrid staining (allows you to see results immediately but take gel picture when the gel is stained more thoroughly)

See Knight:NuPAGE electrophoresis/Gel drying for instructions on how to dry and preserve the gel.


Marker Sizes

  • The SeeBlue Plus 2 marker (blue unless another color is indicated); 10 μL samples have around 1-2 μg of protein per band
    • Myosin 188 KD (MES buffer) 191 KD (MOPS buffer)
    • Phosphorylase B 98 KD (MES buffer) 97 KD (MOPS buffer) (orange)
    • BSA 62 KD (MES buffer) 64 KD (MOPS buffer)
    • Glutamic dehydrogenase 49 KD (MES buffer) 51 KD (MOPS buffer)
    • Alcohol dehydrogenase 38 KD (MES buffer) 39 KD (MOPS buffer)
    • Carbonic anhydrase 28 KD
    • Myoglobin 17 KD (MES buffer) 19 KD (MOPS buffer) (purple)
    • Lysozyme 14 KD
    • Aprotinin 9 KD
    • Insulin B chain 3 KD

Notes

  • 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.
  • There are several ways to screw up this protocol, I list them here in the hopes that it may help people avoid these trivial mistakes.
    1. Failing to remove the seal at the bottom of the precast gel
    2. The inner and outer chambers of running buffer are not sealed from one another.
    3. Reverse the electrodes in the power supply.
    4. Putting the gel in backwards in the apparatus.

Safety

  • Use nitrile gloves when handling acrylamide.
  • Dispose of acrylamide gels and trays as hazardous.

See here for detailed safety information.

References

  1. NuPAGE Technical Guide [NuPAGETechnicalGuide]
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