Introduction

Instructions for electrophoresis of Tris-Glycine Midi Gels are provided below using the XCell4 SureLock™ Midi-Cell. For detailed instructions and use of gels with the Criterion™ Cell, refer to the Novex® Midi Gel System manual available at www.invitrogen.com.

Denaturing Electrophoresis

Prepare Samples

ReagentReduced SampleNon-reduced Sample
Samplex μlx μl
Tris-Glycine SDS Sample Buffer (2X)5 μl5 μl
NuPAGE® Reducing Agent (10X)1 μl--
Deionized Waterto 4 μlto 5 μl
Total Volume10 μl10 μl

Heat samples at 85°C for 2 minutes.

Prepare 1X SDS Buffer

Prepare 1X Tris-Glycine SDS Running Buffer by adding 100 ml 10X Tris- Glycine SDS Running Buffer to 900 ml of deionized water.

Load Sample

Load the appropriate concentration and volume of your protein sample on the gel.

Load Buffer

Fill each Upper Buffer Chamber with 175 ml 1X Tris-Glycine SDS Running Buffer. Fill Lower Buffer Chamber up to the fill line mark with 1X Tris- Glycine SDS Running Buffer.

Run Conditions

Voltage:   125 V constant
Run Time:   105 minutes (dependent on gel percentage)
Expected Current:   40–50 mA/gel (start); 20–25 mA/gel (end)

Non-denaturing (Native) Electrophoresis

Prepare Samples

ReagentNative Sample
Samplex μl
Tris-Glycine Native Sample Buffer (2X)5 μl
Deionized Waterto 5 μl
Total Volume10 μl

Do not heat samples for native electrophoresis.

Prepare 1X Native Buffer

Prepare 1X Tris-Glycine Native Running Buffer by adding 100 ml 10X Tris-Glycine Native Running Buffer to 900 ml of deionized water.

Load Sample

Load the appropriate concentration and volume of your protein sample on the gel.

Load Buffer

Fill each Upper Buffer Chamber with 175 ml of 1X Tris-Glycine Native Running Buffer. Fill Lower Buffer Chamber up to the fill line mark with 1X Tris-Glycine Native Running Buffer

Run Conditions

Voltage:   125 V constant
Run Time:   1–12 hours
Expected Current:   35–40 mA/gel (start); 15–20 mA/gel (end)

License Information

These products are covered by Limited Use Label License No. 5. For details on the license, see the manual supplied with the gel or visit www.invitrogen.com.

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SimplyBlue™ SafeStain Microwave Protocol

SimplyBlue™ SafeStain Microwave protocol for staining Tris-Glycine Midi Gels is described below. For detailed instructions, refer to the Novex® Midi Gel System manual available at www.invitrogen.com.

  1. Place the gel in 150 ml ultrapure water and microwave on high (950-1100 watts).  1 minute

  2. Shake the gel on an orbital shaker. Discard wash. 1 minute

  3. Repeat Steps 1 and 2 twice. 1 minute each

  4. Add 40 ml SimplyBlue™ SafeStain and microwave on high. 45 seconds to 1 minute

  5. Shake the gel on an orbital shaker. Discard stain. 5–10 minutes

  6. Wash the gel in 150 ml ultrapure water on an orbital shaker. 10 minutes

  7. Add 30 ml 20% NaCl and shake the gel on an orbital shaker. 5–10 minutes

  8. Optional: Repeat Step 6 for a clear background. 1 hour

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Western Transfer Protocol

Instructions are provided for blotting Tris-Glycine Midi Gels using a semi-dry blotting apparatus with a lower anode plate. For detailed instructions, refer to the Novex® Midi Gel System manual available at www.invitrogen.com.

Prepare Transfer Buffer

To prepare 500 ml 2X Tris-Glycine Transfer Buffer, mix 40 ml 25X Novex® Tris-Glycine Transfer Buffer, 50 ml methanol, and 410 ml deionized water.

Transferring the Gel Using a Semi-Dry Blotting Apparatus

  1. After removing the gel from the cassette, incubate the gel in 100 ml 2X Tris-Glycine Transfer Buffer (recipe above) for 10 minutes on an orbital shaker.

  2. Soak nitrocellulose blotting membrane in 2X Tris-Glycine Transfer Buffer for a few minutes. For PVDF membrane, wet the PVDF membrane in alcohol (methanol, ethanol, or isopropanol), rinse with deionized water, then soak in 2X Transfer Buffer.

  3. Briefly soak 2 pieces of 2.5 mm thick blotting paper in 2X Tris-Glycine Transfer Buffer. Several pieces of thinner blotting paper can be used to produce a stack of equivalent thickness. Remove any air bubbles between the pape  before use.

  4. Place two pieces of pre-soaked 2.5 mm thick blotting paper (from Step 3) on the anode plate. Remove any air bubbles between the paper and plate with a roller or pipette.

  5. Place the pre-soaked blotting membrane on top of the blotting paper and remove any air bubbles.

  6. Place the gel on top of the membrane and remove any air bubbles with a roller or wet gloved finger. Avoid disturbing the gel after positioning the gel to prevent any protein smearing on the membrane.

  7. Briefly soak the remaining 2 pieces of 2.5 mm thick blotting paper as described in Step 3. Place the 2 pieces of pre-soaked paper on the gel and remove any air bubbles.

  8. Place the cathode plate on the assembly and follow the manufacturer’s instructions to assemble the semi-dry blotting apparatus carefully with minimal disturbance to the gel/membrane assembly.

  9. Transfer at 20 V for 60 minutes.

Note: Depending on the size of your protein and the gel acrylamide concentration used, you may need to optimize the voltage and transfer time after reviewing the initial results.
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25-0913   Version B   12-May-2008