Introduction

Instructions are provided below for electrophoresis of Novex® Zymogram Gels using the XCell SureLock™ Mini-Cell. For detailed instructions, refer to the Novex® Pre-Cast Gel Electrophoresis Guide available at www.invitrogen.com or contact Technical Service.

Prepare Samples


Reagent Sample
Samplex μl
Novex® Tris-Glycine SDS Sample Buffer (2X)5 μl
Deionized Waterto 5 μl
Total Volume10 μl

Do not heat or reduce samples for Zymogram gels.

Prepare Running Buffer

Prepare 1X Tris-Glycine SDS Running Buffer by adding 100 ml of 10X Novex® 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 the Upper Buffer Chamber with 200 ml and the Lower Buffer Chamber with 600 ml of 1X Tris-Glycine SDS Running Buffer.

Run Conditions

Voltage:   125 V constant
Run Time:   90 minutes (dependent on gel percentage)
Expected Current:   30-40 mA/gel (start); 8-12 mA/gel (end)

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Developing the Gel

  1. Dilute Novex® Zymogram Renaturing Buffer (10X) and Novex® Zymogram Developing Buffer (10X), 1:9 with deionized water. You will need 100 ml of each buffer per one or two mini-gels.

  2. After electrophoresis, remove the gel and incubate the gel in 1X Zymogram Renaturing Buffer from Step 1 for 30 minutes at room temperature with gentle agitation.

  3. Decant the Zymogram Renaturing Buffer and add 1X Zymogram Developing Buffer from Step 1 to the gel.

  4. Equilibrate the gel for 30 minutes at room temperature with gentle agitation.

  5. Decant the buffer and add fresh 1X Zymogram Developing Buffer from Step 1 to the gel.

  6. Incubate the gel at 37°C for at least 4 hours or overnight for maximum sensitivity. The optimal result is determined empirically by varying the sample load or incubation time.
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Staining the Gel

Zymogram (Blue Casein) 4-16% gels do not require staining. For nonpre-stained Zymogram gels, stain the gels with Colloidal Blue Staining Kit or the SimplyBlue™ Safestain available from Invitrogen. Areas of protease activity will appear as clear bands against a dark background.
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Sensitivity Level

10% Zymogram (Gelatin) Gel:                     10-6 units of collagenase
12% Zymogram (Casein) Gel:                      7 x 10-4 units of trypsin
4-16% Zymogram (Blue Casein) Gel:        1.5 x 10-3 units of trypsin
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IM-6006B   5-Nov-2004