Introduction

Advantages of Tricine Gels

The Tricine Gels have the following advantages over the Tris-Glycine Gels for resolving proteins in the molecular weight range of 2-20 kDa:

  • Allows resolution of proteins with molecular weights as low as 2 kDa
  • Ideal for direct sequencing of proteins after transferring to PVDF as tricine does not interfere with sequencing
  • Minimizes protein modification as the Tricine buffer system has a lower pH

The Tricine System

The Tricine system is a modification of the Tris-Glycine discontinuous buffer system  specifically designed for the resolution of low molecular weight proteins.

In the Tris-Glycine system, the proteins are stacked in the stacking gel between a highly mobile leading chloride ion (in the gel buffer) and the slower trailing glycine ion (in the running buffer). These stacked protein bands undergo sieving once they reach the separating gel. However, the resolution of smaller proteins (<10 kDa) is hindered by the continuous accumulation of free dodecylsulfate (DS) ions (from the SDS sample and running buffers) in the stacking gel. This zone of stacked DS micelles causes mixing of the DS ions with the smaller proteins resulting in fuzzy bands and decreased resolution. The mixing also interferes with the fixing and staining of smaller proteins.

To solve this problem, the Tricine system uses a low pH of the gel buffer and replaces the trailing glycine ion with a fast moving tricine ion in the running buffer. The smaller proteins that migrate with the stacked DS micelles in the Tris-Glycine system are now well separated from DS ions in the Tricine system resulting in sharper bands and higher resolution.
 
Novex® Tricine Gels are ideal for peptides and low molecular weight proteins (less than 10 kDa). The Tricine Gels are based on the Tricine system developed by (Schaegger and vonJagow, 1987). In this buffer system, tricine substitutes glycine in the running buffer resulting in more efficient stacking and destacking of low molecular weight proteins and higher resolution of smaller peptides. The Novex® Tricine Gels do not contain tricine in the gel, the tricine is supplied by the running buffer. Tricine gels must be used with denatured or reduced proteins only. The separating range of Tricine gels is 2.5-200 kDa.
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Materials Supplied by the User

You will need the following items.

  • Protein sample
  • Deionized water
  • Protein molecular weight markers
  • Tricine SDS Sample Buffer
  • NuPAGE® Reducing Agent for reduced samples
  • Tricine SDS Running Buffer

Storage and Shelf life

Store Novex® Pre-Cast Gels at +4° C. The gels have a shelf life of 4-8 weeks depending upon the gel type when stored at +4° C.

Do not freeze Novex® Pre-Cast Gels.

Use gels immediately from the refrigerator. Extended exposure of the gels to room temperature seriously impairs the performance of the gel.
 
Packaging the Novex® Pre-Cast Gels are supplied as 10 gels per box. Gels are individually packaged in clear pouches with 4-10 ml of Packaging Buffer.

Handling the GelsThe Packaging Buffer contains 0.02% sodium azide and residual acylamide monomer. Wear gloves at all times when handling gels.
Warning:  This product contains a chemical (acrylamide) known to the state of California to cause cancer.
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Ordering Information

Sku Name Size Price Qty
EC6675BOX Novex® 10% Tricine Protein Gels, 1.0 mm, 10 well 1 box USD 215.00
EC6695BOX Novex® 16% Tricine Protein Gels, 1.0 mm, 10 well 1 box USD 217.00
EC6625BOX Novex® 10-20% Tricine Protein Gels, 1.0 mm, 10 well 1 box USD 208.95

Preparing Samples

The Novex® Tricine SDS Sample Buffer (2X) and NuPAGE® Reducing Agent (10X) are available from Invitrogen.

  1. Prepare reduced or non-reduced samples for Tricine gels as described below: Note:  For reduced sample, add the reducing agent immediately prior to electrophoresis to obtain the best results. 


    Reagent Sample Reduced Sample Sample
    Sample x µlx µl
    Novex® Tricine SDS Sample Buffer (2X) 5 µl5 µl
    NuPAGE® Reducing Agent (10X)1 µl--
    Deionized Waterto 4 µlto 5 µl
    Total Volume10 µl10 µl

                                                  

  2. Heat samples at 85° C for 2 minutes. Load the sample immediately on the gel.
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Preparing Running Buffer

Novex® Tricine SDS Running Buffer (10X) is available from Invitrogen.  

  1.   Prepare 1000 ml of 1X Tricine SDS Running Buffer using Novex® Tricine SDS Running Buffer (10X) as follows:    

Novex® Tricine SDS Running Buffer (10X) 100 ml
Deionized Water900 ml
Total Volume1000 ml
                                          
  2.   Heat samples at 85° C for 2 minutes. Load the sample immediately on the gel.

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Electrophoresis of Novex® Pre-Cast Gels

Protocol using XCell SureLock™ Mini-Cell
 
Wear gloves and safety glasses when handling gels

XCell SureLock™ Mini-Cell requires 200 ml for the Upper Buffer Chamber and 600 ml for the Lower Buffer Chamber.

  1. Remove the Novex® Pre-Cast Gel from the pouch.

  2. Rinse the gel cassette with deionized water. Peel off the tape from the bottom of the cassette.

  3. In one smooth motion, gently pull the comb out of the cassette.

  4. Rinse the sample wells with the appropriate 1X SDS Running Buffer. Invert the gel and shake the gel to remove the buffer. Repeat two more times.

  5. Orient the two gels in the Mini-Cell such that the notched “well” side of the cassette faces inwards toward the Buffer Core. Seat the gels on the bottom of the Mini-Cell and lock into place with the Gel Tension Wedge. Refer to the XCell SureLock™ Mini-Cell manual (IM-9003) for detailed instructions. Note: If you are using only one gel, the plastic Buffer Dam replaces the second gel cassette.

  6. Fill the Upper Buffer Chamber with a small amount of the running buffer to check for tightness of seal. If you detect a leak from Upper to the Lower Buffer Chamber, discard the buffer, reseal the chamber, and refill.

  7. Once the seal is tight, fill the Upper Buffer Chamber (inner) with the appropriate 1X running buffer. The buffer level must exceed the level of the wells.

  8. Load an appropriate volume of sample at the desired protein concentration onto the gel.

  9. Load appropriate protein molecular weight markers.

  10. Fill the Lower Buffer Chamber with 600 ml of the appropriate 1X running buffer.

  11. Place the XCell SureLock™ Mini-Cell lid on the Buffer Core. With the power on the power supply turned off, connect the electrode cords to the power supply [red to (+) jack, black to (-) jack].
 
Electrophoresis Conditions

Run your gels according to the following protocol:  

Gel Type
Voltage
Expected Current*
Run Time
Tricine Gels
125 V constant
Start: 80 mA
End: 40 mA
90 minutes (dependent on gel type)
Run the gel until the phenol red tracking dye reaches the bottom of the gel.

*Expected start and end current values are stated for single gels.

 
Removing the Gel after Electrophoresis

  1. After electrophoresis is complete, shut off the power, disconnect electrodes, and remove gel(s) from the XCell SureLock™ Mini-Cell.

  2. Separate each of the three bonded sides of the cassette by inserting the Gel Knife into the gap between the cassette’s two plates. The notched (“well”) side of the cassette should face up.

  3. Push down gently on the knife handle to separate the plates. Repeat on each side of the cassette until the plates are completely separated. Caution: Use caution while inserting the gel knife between the two plates to avoid excessive pressure towards the gel.

  4. Carefully remove and discard the top plate, allowing the gel to remain on the bottom (slotted) plate.

  5. If blotting, proceed without removing the gel from the bottom plate.

  6. If staining, remove the gel from the plate by one of the methods:

  • Use the sharp edge of the gel knife to remove the bottom lip of the gel. The gel knife should be at a 90° angle, perpendicular to the gel and the slotted half of the cassette. Push down on the knife, and then repeat the motion across the gel to cut off the entire lip. Hold the plate and gel over a container with the gel facing downward and use the knife to carefully loosen one lower corner of the gel and allow the gel to peel away from the plate.
  • Hold the plate and gel over a container with the gel facing downward. Gently push the gel knife through the slot in the cassette, until the gel peels away from the plate. Cut the lip off of the gel after fixing, staining, but before drying.
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Troubleshooting

Introduction

Review the information below to troubleshoot your experiments with Novex® Gels.

Problem
Cause
Solution
Run taking longer time
Running buffer too dilute
Make fresh running buffer as described in this manual and avoid adjusting the pH of the 1X running buffer.
Low or no current during the run
Incomplete circuit
  • Remove the tape from the bottom of the cassette prior to electrophoresis.
  • Make sure the buffer covers the sample wells.
  • Check the wire connections on the buffer core to make sure the connections are intact.
Faint shadow or “ghost” band below the expected protein band
Ghost bands are caused due to a slight lifting of the gel from the cassette resulting in trickling of some sample beyond its normal migration point. Gel lifting off the cassette is caused due to:
  • Expired gels
  • Improper storage of gels
 
 
 
 
 
  • Avoid using expired gels. Use fresh gels
  • ·Store the gels at the appropriate temperature.
Streaking of proteins
  • Sample overload
  • High salt concentration in the sample
  • Sample precipitates
  • Contaminants such as membranes or DNA complexes in the sample
  • Load the appropriate amount of protein
  • Decrease the salt concentration of your sample using dialysis or gel filtration
  • Increase the concentration of SDS in your sample if necessary, to maintain the solubility of the protein.
  • Centrifuge or clarify your sample to remove particulate contaminants
Bands in the outer lane of the gel are curving upwards
  • Concentrated buffer used
  • Expired gels used
 
  • The pre-made buffers are supplied as concentrate. Dilute the buffers as described in this manual.
  • Avoid using gels after the expiration date.
Bands in the outside lanes of the gel “smiling”
Expired gels used causing the acrylamide to break down in the gel
Avoid using gels after the expiration date. Use fresh gels.
Bands are running as U shape rather than a flat band
Samples are loaded on the gel and not electrophoresed immediately resulting in sample diffusion
Load samples on to the gel immediately before electrophoresis.
Bands appear to be “funneling” or getting narrower as they progress down the gel
Proteins are over-reduced causing the proteins to be negatively charged and repel each other.
Reduce the proteins using DTT or ß-mercaptoethanol.
Dumbbell shaped bands after electrophoresis
Loading a large volume of sample causing incomplete stacking of the entire sample. This effect is intensified for larger proteins
Load the appropriate volume of sample per well. If your sample is too dilute, concentrate the sample using salt precipitation or ultrafiltration.
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References

  1. Kubo, K. (1995). Effect of Incubation of Solutions of Proteins Containing Dodecyl Sulfate on the Cleavage of Peptide Bonds by Boiling. Anal. Biochem. 225, 351-353.

  2. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

  3. Ornstein, L. (1964). Disc Electrophoresis, 1, Background and Theory. Ann New York Acad. Sci 121, 321-349.

  4. Revzin, A. (1989). Gel Electrophoresis Assays for DNA-Protein Interactions. BioTechniques 4, 346-355.

  5. Schaegger, H., and vonJagow, G. (1987). Tricine-Sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis for the Separation of Proteins in the Range from 1 to 100 kDa. Anal. Biochem. 166, 368-379.
 
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