protocol

Introduction

The Tris-Glycine gels are based on the Laemmli System (Laemmli, 1970) with minor modifications for maximum performance in the pre-cast format. The separating and stacking gels of Novex® Tris-Glycine gels have a pH of 8.65 unlike traditional Laemmli gels that have a stacking gel pH of 6.8 and separating gel pH of 8.8. The separating range of Tris-Glycine gels is 6-200 kDa.
 
Tris-Glycine Discontinuous Buffer System
 
The Tris-Glycine discontinuous buffer systems involves three ions:

  • Chloride (-) is supplied by the gel buffer and serves as a leading ion due to its high affinity to the anode as compared to other anions in the system. The gel buffer ions are Tris+ and Cl- (pH 8.65).
  • Glycine (-) is the primary anion supplied by the running buffer and serves as a trailing ion. Glycine is partially negatively charged and trails behind the highly charged chloride ions in the charged environment. The running buffer ions are Tris+, Gly-, and dodecylsulfate- (pH 8.3).
  • Tris Base (+) is the common ion present in the gel buffer and running buffer. During electrophoresis, the gel and buffer ions in the Tris-Glycine system form an operating pH of 9.5 in the separation region of the gel. 

TOP

Materials and Storage

Materials Supplied by the User
 
You will need the following items. Recipes are provided on Recipes section if you are preparing your own buffers.

  • Protein sample
  • Deionized water
  • Protein molecular weight markers

For denaturing electrophoresis

  • Tris-Glycine SDS Sample Buffer
  • NuPAGE® Reducing Agent
  • Tris-Glycine SDS Running Buffer

For non-denaturing electrophoresis

  • Tris-Glycine Native Sample Buffer
  • Tris-Glycine Native 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 Gels

The 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. Refer to the MSDS.
TOP

Protocol - Electrophoresis of Novex® Tris-Glycine Pre-Cast Gels

Introduction

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). You may download this manual from our website or contact Technical Service.

If you are using any other mini-cell for electrophoresis, refer to the manufacturer’s recommendations.
 
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 (see Recommended loading volumes).

  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].


See below for Electrophoresis Conditions.
 
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


*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.


  7. Fix and stain the gel as described.

Preparing Sample

The Novex® Tris-Glycine SDS or Native Sample Buffer (2X) and NuPAGE® Reducing Agent (10X) are available from Thermo Fisher Scientific.

  1. Prepare denatured or non-denatured (native) samples for Tris-Glycine gels as described below: Note: For reduced samples, add the reducing agent to a final concentration of 1X immediately prior to electrophoresis to obtain the best results.

  2. Reagent Denatured Sample Native Sample
    Sample x µl x µl
    Tris-Glycine SDS Sample Buffer (2X) 5 µl --
    Tris-Glycine Native Sample Buffer (2X) --  
    Deionized Water to 5 µl to 5 µl
    Total Volume 10 µl 10 µl


  3. Heat the sample at 85° C for 2 minutes. Load the sample immediately on the gel. Do not heat samples for native electrophoresis.



Running Reduced and Non-Reduced Samples

For optimal results, we do not recommend running reduced and non-reduced samples on the same gel.
If you do choose to run reduced and non-reduced samples on the same gel, do not run reduced and non-reduced samples in adjacent lanes. The reducing agent may have a carry-over effect on the non-reduced samples if they are in close proximity.
 
Heating Samples

Heating the sample at 100° C in SDS containing buffer results in proteolysis (Kubo, 1995). We recommend heating samples for denaturing electrophoresis (reduced or non-reduced) at 85°C for 2-5 minutes for optimal results.
Do not heat the samples for non-denaturing (native) electrophoresis.
 
Recommended Buffers

The recommended running buffer and sample buffer for each Novex® Pre-Cast Gel is listed in the table below. You will prepare your sample in the appropriate sample buffer so the final concentration of the sample buffer will be 1X. You will need to dilute the running buffer to 1X final concentration before use.
See recipes if you are making your own buffers.

Gel Type Running Buffer Sample Buffer
Tris-Glycine Gel (SDS-PAGE) Tris-Glycine SDS Running Buffer (10X) Tris-Glycine SDS Sample Buffer (2X)


  
Reducing Agent

The NuPAGE® Reducing Agent contains 500 mM dithiothreitol (DTT) at a 10X concentration in a ready-to-use, stabilized liquid form and is used to prepare samples for reducing gel electrophoresis.

ß-mercaptoethanol can be used with the Novex® gels at a final concentration of 2.5%. Choice of the reducing agent is a matter of preference and either DTT or ß-mercaptoethanol can be used. We recommend adding the reducing agent to the sample within an hour of loading the gel.

Avoid storing reduced samples for long periods even if they are frozen. This will result in the reoxidation of samples during storage and produce inconsistent results.
 
Running Reduced and Non-Reduced Samples

For optimal results, we do not recommend running reduced and non-reduced samples on the same gel.
If you do choose to run reduced and non-reduced samples on the same gel, do not run reduced and non-reduced samples in adjacent lanes. The reducing agent may have a carry-over effect on the non-reduced samples if they are in close proximity.

Preparing Running Buffer

Novex® Tris-Glycine SDS or Native Running Buffer (10X) is available from Thermo Fisher Scientific.

  1.   Prepare 1000 ml of 1X Tris-Glycine SDS or Native Running Buffer using Novex® Tris-Glycine SDS or Native Running Buffer (10X) as follows:

Novex® Tris-Glycine SDS or Native Running Buffer (10X) 100 ml
Deionized Water 900 ml
Total Volume 1000 ml


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

See recipes for the Novex® Tris-Glycine SDS and Native Running Buffers, if you are preparing the running buffers

Calibrating Protein Molecular Weight

Introduction

The molecular weight of a protein can be determined based upon its relative mobility by constructing a standard curve with protein standards of known molecular weights.

The protein mobility in SDS-PAGE gels is dependent on the:

  • Length of the protein in its fully denatured state
  • SDS-PAGE buffer systems
  • Secondary structure of the protein


An identical molecular weight standard may have slightly different mobility resulting in different apparent molecular weight when run in different SDS-PAGE buffer systems.  If you are using the Novex® protein molecular weight standards, see the apparent molecular weights of these standards on the Novex® Pre-Cast Gels listed to determine an apparent molecular weight of your protein.
 
Protein Secondary Structure

When using SDS-PAGE for molecular weight determination, slight deviations from the calculated molecular weight of a protein (calculated from the known amino acid sequence) can occur due to the retention of varying degrees of secondary structure in the protein, even in the presence of SDS. This phenomenon is observed in highly organized secondary structures (such as collagens, histones, or highly hydrophobic membrane proteins) and in peptides, where the effect of local secondary structure and amino acid side chains becomes magnified relative to the total size of the peptide.
 
Buffer Systems

Slight differences in protein mobilities also occur when the same proteins are run in different SDS-PAGE buffer systems. Each SDS-PAGE buffer system has a different pH, which affects the charge of a protein and its binding capacity for SDS. The degree of change in protein mobility is usually small in natural proteins but more pronounced with “atypical” or chemically modified proteins such as pre-stained standards.
 
Assigned Apparent Molecular Weights

The apparent molecular weight values currently provided with the Novex® molecular weight standards were derived from the construction of a calibration curve in the Tris-Glycine SDS-PAGE System. We have now calculated and assigned apparent molecular weights for the Novex® protein standards in several buffer systems. Remember to use the one that matches your gel for the most accurate calibration of your protein.
The following charts summarize the approximate molecular weight values for the Novex® protein molecular weight standards when run in different buffer systems. You may generate calibration curves in your lab with any other manufacturer’s standards.

MultiMark® Multi-Colored Standard
Tris-Glycine Gels (4-20%)
Myosin
250 kDa
Phosphorylase B
148 kDa
Glutamic Dehydrogenase
60 kDa
Carbonic Anhydrase
42 kDa
Myoglobin (Blue)
30 kDa
Myoglobin (Red)
22 kDa
Lysozyme
17 kDa
Aprotinin
6 kDa
Insulin
4 kDa

 

Mark 12 Unstained Standard
Tris-Glycine Gels (4-20%)
Myosin
200 kDa
ß-Galactosidase
116.3 kDa
Phosphorylase B
97.4 kDa
Bovine Serum Albumin
66.3 kDa
Glutamic Dehydrogenase
55.4 kDa
Lactate Dehydrogenase
36.5 kDa
Carbonic Anhydrase
31 kDa
Trypsin Inhibitor
21.5 kDa
Lysozyme
14.4 kDa
Aprotinin
6 kDa
Insulin B Chain
Unresolved Insulin

 

SeeBlue® Pre-Stained Standard
 
Tris-Glycine Gel (4-20%)
Myosin
250 kDa
BSA
98 kDa
Glutamic Dehydrogenase
64 kDa
Alcohol Dehydrogenase
50 kDa
Carbonic Anhydrase
36 kDa
Myoglobin
30 kDa
Lysozyme
16 kDa
Aprotinin
6 kDa
Insulin
4 kDa

 

SeeBlue® Plus2 Pre-Stained Standard
 
Tris-Glycine Gel (4-20%)
Myosin
250 kDa
Phosphorylase B
148 kDa
BSA
98 kDa
Glutamic Dehydrogenase
64 kDa
Alcohol Dehydrogenase
50 kDa
Carbonic Anhydrase
36 kDa
Myoglobin
22 kDa
Lysozyme
16 kDa
Aprotinin
6 kDa
Insulin
4 kDa

 

TOP

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
  • Load the appropriate amount of protein as described.
 
  • High salt concentration in the sample
  • Decrease the salt concentration of your sample using dialysis or gel filtration
 
  • Sample precipitates
  • Increase the concentration of SDS in your sample if necessary, to maintain the solubility of the protein.
 
  • Contaminants such as membranes or DNA complexes in the sample
  • Centrifuge or clarify your sample to remove particulate contaminants
Bands in the outer lane of the gel are curving upwards
  • Concentrated buffer used
  • The pre-made buffers are supplied as concentrate. Dilute the buffers as described in this manual.
 
  • Expired gels used
  • Avoid using gels after the expiration date.
 
  • High voltage used
  • 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 ß-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.

Recipes

Tris-Glycine SDS Running Buffer

The Tris-Glycine SDS Running Buffer is available from Thermo Fisher Scientific.

25 mM Tris Base
192 mM Glycine
0.1% SDS
pH 8.3

  1.   To prepare 1000 ml of 10 X Tris-Glycine SDS Running Buffer, dissolve the following reagents to 900 ml ultrapure water:

Tris Base                                             29 g
Glycine                                               144 g
SDS                                                      10 g

  2.   Mix well and adjust the volume to 1000 ml with ultrapure water.

  3.   Store at room temperature. The buffer is stable for 6 months when stored at room temperature.

  4.   For electrophoresis, dilute this buffer to 1X with water. The pH of the 1X solution is 8.3. Do not use acid or base to adjust the pH.
 
Tris-Glycine Native Running Buffer

The Tris-Glycine Native Running Buffer is available from Thermo Fisher Scientific.

25 mM Tris base
192 mM Glycine
pH 8.3

  1.   To prepare 1000 ml of 10 X Tris-Glycine Native Running Buffer, dissolve the following reagents to 900 ml ultrapure water:

Tris Base                                             29 g
Glycine                                               144 g

  2.   Mix well and adjust the volume to 1000 ml with ultrapure water.

  3.   Store at room temperature. The buffer is stable for 6 months when stored at room temperature.

  4.   For native electrophoresis, dilute this buffer to 1X with water. The pH of the 1X solution is 8.3. Do not use acid or base to adjust the pH.
 
Tris-Glycine SDS Sample Buffer

The Tris-Glycine SDS Sample Buffer is available from Thermo Fisher Scientific.


63 mM Tris HCl
10% Glycerol
2% SDS
0.0025% Bromophenol Blue
pH 6.8

  1.   To prepare 10 ml of 2X Tris-Glycine SDS Sample Buffer, mix the following reagents :


0.5 M Tris-HCl, pH 6.8                                 2.5 ml
Glycerol                                                         2 ml
10% (w/v) SDS                                             4 ml
0.1% (w/v) Bromophenol Blue                   0.5 ml

  2.   Adjust the volume to 10 ml with ultrapure water.

  3.   Store at +4° C. The buffer is stable for 6 months when stored at +4° C.
 
Tris-Glycine Native Sample Buffer

The Tris-Glycine Native Sample Buffer is available from Thermo Fisher Scientific.

100 mM Tris HCl
10% Glycerol
0.0025% Bromophenol Blue
pH 8.6

  1.   To prepare 10 ml of 2X Tris-Glycine Native Sample Buffer, mix the following reagents :
 
0.5 M Tris HCl, pH 8.6                                    4 ml
Glycerol                                                         2 ml
0.1% (w/v) Bromophenol Blue                   0.5 ml

  2.   Adjust the volume to 10 ml with ultrapure water.

  3.   Store at +4° C. The buffer is stable for 6 months when stored at +4° C.
 
Tris-Glycine Transfer Buffer

The Tris-Glycine Transfer Buffer is available from Thermo Fisher Scientific.

12 mM Tris Base
96 mM Glycine
pH 8.3

  1.   To prepare 500 ml of 25 X Tris-Glycine Transfer Buffer, dissolve the following reagents in 400 ml ultrapure water:

Tris Base                                                           18.2 g
Glycine                                                                 90 g

  2.   Mix well and adjust the volume to 500 ml with ultrapure water.

  3.   Store at room temperature. The buffer is stable for 6 months when stored at room temperature.

  4.   For blotting, dilute this buffer as described. The pH of the 1X solution is 8.3. Do not use acid or base to adjust the pH. 

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.
TOP

Appendix

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 for 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.

Specifications
 
Gel Matrix:                                             Acrylamide/Bisacrylamide
Gel Thickness:                                      1.0 mm or 1.5 mm
Gel Size:                                                 8 cm x 8 cm
Cassette Size:                                       10 cm x 10 cm
Cassette Material:                                Styrene Copolymer (recycle code 7)
Sample Well Configuration                 1, 5, 9, 10, 12, 15-well, 2D -well, and IPG well
 
Recommended Loading Volumes
The recommended loading volumes and protein load per band by the detection method are provided in the table below.

Note:  The 9-well gels are compatible with any eight-channel pipettors used for loading samples from 96-well plates. An additional lane is included for loading protein molecular weight standard. 

TOP
LT058