Introduction

A large variety of pre-cast gels are available from Invitrogen.  These include gels for analysis of proteins (Tris-Glycine, Tricine, Zymogram, IEF, and ZOOM® Gels) and nucleic acids (TBE, TBE-Urea, and DNA Retardation).   General information on Novex® Pre-Cast Gels is provided in this section.
 
Novex® Pre-Cast Gels are capable of resolving proteins in the range of 2-500 kDa and nucleic acids in the range of 10-3000 bp.

Gel-Shift Assay

The gel shift assay is based on the fact that the movement of a DNA molecule through a non-denaturing polyacrylamide gel is hindered when bound to a protein molecule (Revzin, 1989). This technique is used to characterize DNA/protein complexes. Detection is performed with ethidium bromide staining of DNA or, for greater sensitivity, with radiolabeling the DNA or protein.
 
Choosing a Gel for Your Application

To obtain the best results for your application, it is important to choose the correct gel percentage, buffer system, gel format, and thickness.   A variety of factors affect the choice of a gel. These include:
 
Application

Based on the type of your application, you can choose from gels for protein separation (Tris-Glycine, Tricine, IEF, ZOOM®, and Zymogram Gels) or gels for nucleic acid separation (TBE, TBE-Urea, and DNA Retardation Gels).
 
Size of the molecule being separated

Large molecules resolve well on a low percentage gels while small molecules are best resolved on high percentage gels.  The size of the molecule usually dictates the acrylamide percentage.  If you do not know the molecular weight of the molecule or are separating a wide molecular weight range of molecules, choose gradient gels.
 
Amount of available material

The higher the number of wells and the thinner the gel, the lower the sample loading volume and vice versa.  Based on the amount of your starting material available, you can choose from a variety of comb types.

Note:  Proteins will transfer more easily out of a 1.0 mm gel than a 1.5 mm gel.
 
Refer to the Gel Migration Chart on our Web site at www.invitrogen.com or in our catalog to choose the right gel for your application. Choose a gel such that the molecules migrate about 70% of the length of gel for best resolution (gray shaded area on the Gel Migration Chart).
 
Compatibility

The size of a Novex® Pre-Cast Gel is 10 x 10 cm (gel size is 8 x 8 cm).  We recommend using the XCell SureLock™ Mini-Cell or the electrophoresis of Novex® Pre-Cast Gels to obtain optimal and consistent performance.  Novex® Pre-Cast Gels are compatible with most other mini-cells designed for electrophoresis of 10 cm (h) x 10 cm (w) gel cassettes.
 
Staining Novex® Pre-Cast Gels

The Novex® Pre-Cast Gels are compatible with most silver staining protocols.  We recommend using the SilverQuest™ Silver Staining Kit or the SilverXpress® Silver Staining Kit for silver staining of Novex® Gels.
 
The Novex® Pre-Cast Gels are compatible with any of the standard Coomassie® staining procedures.  The protocols that are accelerated by heat are preferable as heat serves as a “fix” for proteins, especially smaller peptides.  The SimplyBlue™ SafeStain and Novex® Colloidal Coomassie® Blue Staining Kit are recommended for staining Novex® Gels.
 
Applications
 
  • Separating proteins over a wide range of molecular weights:  The Novex® Tris-Glycine Gels are used for separating proteins over a wide molecular weight range under denaturing or non-denaturing conditions.
  • Resolving low molecular weight proteins and peptides:  The Novex® Tricine Gels provide high resolution of low molecular weight proteins and peptides.
  • Performing Isoelectric focusing (IEF):  Use Novex® IEF Gels for native (vertical) IEF of proteins.
  • Detecting Proteases:  The Novex® Zymogram Gels are used for detecting and characterizing proteases that utilize casein or gelatin as the substrate.
  • Performing 2D Separation of Proteins:  The ZOOM® Gels are specifically designed for second dimension electrophoresis of 7.0 cm IPG strips.
  • Performing Nucleic Acid Analysis:  The Novex® TBE Gels are used to analyze DNA fragments including restriction digest, PCR products, Southern analysis, and primer analysis. The Novex® TBE-Urea Gels are used for denaturing nucleic acid analysis and are suited for RNase Protection Assays, in-vitro transcription studies, RNA stability studies, and oligonucleotide purification.
  • Performing Gel Shift Assays:  The Novex® 6% DNA Retardation Gels are used to perform gel shift assays.
  • DNA Retardation Gels:  Novex® DNA Retardation Gels consist of 6% polyacrylamide prepared with 1/2X TBE as the gel buffer. The 6% gel provides good resolution of fragments in the range of 60-2500 bp used for DNA retardation assays. The 1/2X TBE buffer is sufficient for good electrophoretic separation yet low enough to promote DNA/ protein interactions. 

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Materials

You will need the following items:

  • DNA sample
  • Deionized water
  • Hi-Density TBE Sample Buffer
  • TBE Running Buffer

Specific buffer conditions may be required during incubation of the protein and DNA target sequence in order to minimize non-specific DNA/protein interactions for certain samples.

If salt concentration is low (0.1 M or less), the samples can usually be loaded in the incubation buffer after adding about 3-5% glycerol and a small amount of bromophenol blue tracking dye.
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Ordering Information

Sku Name Size Price Qty
EC6365BOX DNA Retardation Gels (6%), 1.0 mm, 10 well 1 box USD 139.00
EC63652BOX DNA Retardation Gels (6%), 1.0 mm, 12 well 1 box USD 139.00
EC63655BOX DNA Retardation Gels (6%), 1.0 mm, 15 well 1 box USD 139.00

Protocol - Electrophoresis of Novex® Pre-Cast Gels

Instructions are provided below for electrophoresis of the Novex® Pre-Cast Gels using the XCell SureLock™ Mini-Cell.  For more information on the XCell SureLock™ Mini-Cell, refer to the manual (IM-9003).

Preparing Samples

  1. Prepare samples for DNA Retardation Gels as described below:


Reagent Amount
Sample x µl
Novex® Hi-Density TBE Sample Buffer (5X) 1 µl
Deionized Water to 9 µl
Total Volume 10 µl

  1. Load the samples immediately on the gel. Preparing Running Buffer Novex® TBE Running Buffer (5X).

  2. Prepare 1000 ml of 1/2X (0.5X) TBE Running Buffer using Novex® TBE Buffer (5X) as follows:


Novex® TBE Running Buffer (5X)  100 ml
Deionized Water  900 ml
Total Volume 1000 ml

  1. Mix thoroughly. Use this buffer to fill the Upper and Lower Buffer Chamber of the XCell SureLock™ Mini-Cell for electrophoresis.

 
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.
 

  1. 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.

  2. 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.

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

  4. Load appropriate protein molecular weight markers.

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

  6. 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
Tris-Glycine Gels (SDS-PAGE)
125 V constant
Start: 30-40mA
End: 8-12 mA
90 minutes (dependent on gel type)
Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
Tris-Glycine Gels (Native-PAGE)
125 V constant
Start: 6-12 mA
End: 3-6 mA
1-12 hours
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.
Zymogram Gels
125 V constant
Start: 30-40 mA
End: 8-12 mA
90 minutes (dependent on gel type)
Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
IEF Gels
100 V constant-1 hour
200 V constant-1 hour
500 V constant-30 min
Start: 5 mA
End: 6 mA
2.5 hours
TBE Gels
200 V constant**
Start: 10-18 mA
End: 4-6 mA
30-90 minutes (dependent on gel type)
Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
6% TBE-Urea Gels
180 V constant**
Start: 19 mA
End: 14 mA
50 minutes
Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
10% TBE-Urea Gels
180 V constant**
Start: 15 mA
End: 8 mA
60 minutes
Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
15 % TBE-Urea Gels
180 V constant**
Start: 13 mA
End: 6 mA
75 minutes
Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.
DNA Retardation Gels
100 V constant
Start: 12-15 mA
End: 6-15 mA
90 minutes
Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel.

*Expected start and end current values are stated for single gels.
**Voltages up to 250 V may be used to reduce the run time.

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.
 

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

  2. 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.
  1. Fix and stain the gel.

Troubleshooting

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 as described
  • 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
  • High voltage used

 

  • The pre-made buffers are supplied as concentrate. Dilute the buffers as described in this manual.
  • Avoid using gels after the expiration date.
  • Electrophorese the gel using conditions described.

 

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 b-mercaptoethanol as described.
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 as described. If your sample is too dilute, concentrate the sample using salt precipitation or ultrafiltration.
For TBE-Urea gels
High background and smeared bands or abnormal band shapes
  • RNase contamination
  • Sample renatured
  • Sample overloaded
  • Urea not completely flushed from the wells
  • Always wear gloves and use sterile techniques to prevent RNase contamination.
  • Heat the sample for 3 minutes at 70°C and keep the sample in ice to prevent renaturation. Proceed to electrophoresis immediately after loading.
  • Recommended DNA load is 0.16-0.33 µg/band.
  • Be sure to thoroughly flush urea out of the wells prior to loading the sample.
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