One-Dimensional SDS Gel Electrophoresis of Proteins with NuPAGE® Novex® Pre-Cast Gels

• Novex® Protein Separation and Blotting
• NuPAGE
• MagicMark
  

• Hydrolysis of polyacrylamide at the high gel casting pH of 8.7 resulting in a short shelf life of 4-6 weeks
• Chemical modifications such as deamination and alkylation of proteins due to the high pH (9.5) of the separating gel
• Reoxidation of reduced disulfides from cysteine containing proteins as the redox state of the gel is not constant
• Cleavage of Asp-Pro bond of the proteins when heated at 100° C in the Laemmli sample buffer, pH 5.2 (Kubo, 1995).

• Longer shelf life of 8-12 months due to improved gel stability 
• Improved protein stability during electrophoresis at neutral pH resulting in sharper band resolution and accurate results (Moos et al, 1998)
• Complete reduction of disulfides under mild heating conditions (70° C for 10 minutes) and absence of cleavage of asp-pro bonds using the NuPAGE® LDS Sample buffer (pH > 7.0 at 70° C)
• Reduced state of the proteins maintained during electrophoresis and blotting of the proteins by the NuPAGE® Antioxidant

NuPAGE® Electrophoresis System Components

The NuPAGE® Electrophoresis System consists of:

• NuPAGE® Novex Bis-Tris [Bis (2-hydroxyethyl) imino-tris (hydroxymethyl) methane-HCl] Pre-Cast Gels for separating small to mid-size molecular weight proteins
• NuPAGE® Novex Tris-Acetate Pre-Cast Gels for separating large molecular weight proteins
• NuPAGE® LDS (Lithium dodecyl sulfate) Sample Buffer
• NuPAGE® Reducing Agent
• NuPAGE® Antioxidant
• NuPAGE® MES [2-(N-morpholino) ethane sulfonic acid] SDS or MOPS [3-(N-morpholino) propane sulfonic acid] SDS Running Buffer for NuPAGE® Novex Bis-Tris Gels
• NuPAGE® Tris-Acetate SDS Running Buffer for NuPAGE® Novex Tris- Acetate Gels
• NuPAGE® Transfer Buffer for blotting of NuPAGE® Novex Pre-Cast Gels

 
NuPAGE® Bis-Tris Discontinuous Buffer System

The NuPAGE® Bis-Tris discontinuous buffer system 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 Bis-Tris+ and Cl- (pH 6.4).
• MES or MOPS (-) serves as the trailing ion. The running buffer ions are Tris+, MOPS-/MES-, and dodecylsulfate (-) (pH 7.3-7.7).
• Bis-Tris (+) is the common ion present in the gel buffer and running buffer. The combination of a lower pH gel buffer (pH 6.4) and running buffer (pH 7.3-7.7) results in a significantly lower operating pH of 7 during electrophoresis.

 
NuPAGE® Tris-Acetate Discontinuous Buffer System

The NuPAGE® Tris-Acetate discontinuous buffer system involves three ions:

• Acetate (-) 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 Acetate- (pH 7.0).
• Tricine (-) serves as the trailing ion from the running buffer. The running buffer ions are Tris+, Tricine-, and dodecylsulfate (-) (pH 8.3).
• Tris (+) is the common ion present in the gel buffer and running buffer. The Tris-Acetate system also operates at a significantly lower operating pH of 8.1 during electrophoresis.

 
Separation Range of Proteins

The NuPAGE® Gels have a wider range of separation on a single gel and also separate proteins evenly throughout the low and high molecular weight ranges than existing gels. Due to these advantages, most proteins are well resolved on one of the five NuPAGE® gels.  By combining any of the NuPAGE® Novex Bis-Tris Gels with the MES SDS or MOPS SDS Running Buffer, you can obtain six separation ranges for resolving proteins over a wide molecular weight range of 1-200 kDa. The NuPAGE® Novex Tris-Acetate gels resolve proteins in the molecular weight range of 36-400 kDa. To choose the correct NuPAGE® Gel for your application, refer to the Gel Conversion Guide on our website or the catalog.
 
Types of NuPAGE® Gels

The NuPAGE® Novex Pre-Cast Gels are available in different acrylamide concentrations, gel thickness, and well formats (see the table below).
  

Formulation

The formulation for the NuPAGE® Gels is listed below:

The NuPAGE® Gels do not contain SDS. However, they are designed for performing denaturing gel electrophoresis.
 
Crosslinker

The crosslinker concentration for the NuPAGE® Novex Pre-Cast Gel ranges from 3.8-5% depending on the region of the gel.

Compatibility

The size of a NuPAGE® 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 NuPAGE® Novex Pre-Cast Gels to obtain optimal and consistent performance.
 
Staining NuPAGE® Gels

The NuPAGE® 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 NuPAGE® Gels. The NuPAGE® Novex Pre-Cast Gels are compatible with any of the standard Coomassie staining procedures. The protocols that are accelerated by heat are preferable as the heat serves as a “fix” for proteins, especially smaller peptides. The SimplyBlue™ SafeStain and Novex® Colloidal Coomassie Blue Staining Kit  are recommended for staining NuPAGE® Gels. The NuPAGE® Novex Pre-Cast Gels are also compatible with copper or zinc staining.
 
Applications

The NuPAGE® Novex Pre-Cast Gels are used:

• For separating proteins under denaturing conditions (NuPAGE® Bis-Tris Gels and NuPAGE® Tris-Acetate Gels)
• For separating proteins under non-denaturing (native) conditions (NuPAGE® Tris-Acetate Gels).
• For protein sequencing using Edman sequencing (from gels or PVDF)

Note:   Do not use the NuPAGE® Bis-Tris Gels with NuPAGE® MOPS or MES Running Buffer without SDS for native gel electrophoresis. This buffer system may generate excessive heat resulting in poor band resolution. The protein of interest may not migrate very well in a neutral pH environment if it is not charged.
 
Storage and Shelf life

Store NuPAGE® Novex Bis-Tris Gels at 4-25° C and NuPAGE® Novex Tris-Acetate Gels at +4° C.

The NuPAGE® Novex Bis-Tris Gels have a shelf life of 12 months when stored at 4-25° C.

The NuPAGE® Novex Tris-Acetate Gels have a shelf life of 8 months when stored at 4° C.
 
Do not freeze NuPAGE® Gels.

Using expired gels or improperly stored gels may result in poor band resolution.
 
NuPAGE® Gel Specifications
 
Specifications

Gel Matrix:                                            Acrylamide/Bisacrylamide
Gel Thickness:                                    1.0 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, 9, 10, 12, 15, 17-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- and 17-wells are compatible with any eight-channel pipette used for loading samples from 96-well plates. An additional lane is included for loading protein molecular weight standard.

ZOOM® Gels are 8 x 8 cm, 1.0 mm thick pre-cast polyacrylamide gels cast in a 10 x 10 cm cassette. The ZOOM® Gels are used for 2D analysis of proteins following isoelectric focusing of 7.0 cm IPG strips. ZOOM® Gels contain an IPG well and a molecular weight marker well. The IPG well is designed to accommodate a 7.0 cm IPG strip.

Two types of ZOOM® Gels are available

• NuPAGE® Novex 4-12% Bis-Tris ZOOM® Gel


• Novex® 4-20% Tris-Glycine ZOOM® Gel

 
Second Dimension Electrophoresis

The second dimension electrophoresis procedure involves reducing and alkylating the proteins focused on your IPG strip in equilibration buffer, loading the strip on your second dimension gel, and performing SDS-PAGE.
 
Before Starting

You will need the following items:

• 4X NuPAGE® LDS Sample Buffer


• NuPAGE® Sample Reducing Agent·        
• NuPAGE® Novex 4-12% Bis-Tris ZOOM® Gel or Novex® 4-20% Tris-Glycine ZOOM™ Gel 
• Appropriate running buffer depending on the type of gel you are using
• 0.5% agarose solution
• Iodoacetamide
• Plastic flexible ruler or thin weighing spatula
• 15 ml conical tubes
• Water bath set at 55° C or 65° C
• XCell SureLock™ Mini-Cell
• Protein molecular weight marker  

Equilibrating the IPG Strip

1. Dilute 4X NuPAGE® LDS Sample Buffer to 1X with deionized water.


2. Add 500 µl of the NuPAGE® Sample Reducing Agent to 4.5 ml of the 1X NuPAGE® LDS Sample Buffer from Step 1 in a 15 ml conical tube. Place one IPG strip in this conical tube for equilibration.


3. Incubate for 15 minutes at room temperature. Decant the Reducing Solution.


4. Prepare 125 mM Alkylating Solution fresh by adding 116 mg of iodoacetamide to 5 ml of 1X NuPAGE® LDS Sample Buffer from Step 1.


5. Add 5 ml of Alkylating Solution (from Step 4) to the conical tube containing the IPG strip. Incubate for 15 minutes at room temperature.


6. Decant the Alkylating Solution and proceed to SDS-PAGE. Use the equilibrated IPG strip immediately for second dimension.

 
SDS-PAGE

A protocol for SDS-PAGE is provided below using ZOOM® Gels with the XCell SureLock™ Mini-Cell. You may download the XCell SureLock™ Mini-Cell manual from our website or contact Technical Service. If you are using any other electrophoresis system, refer to the manufacturer’s recommendations.

1. Prepare 0.5% agarose solution in the appropriate running buffer and keep it warm (55-65° C) until you are ready to use the agarose solution.


2. If the molecular weight marker well is bent, straighten the well using a gel loading tip.


3. Cut the plastic ends of the IPG strip flush with the gel. Do not cut off any portions of the gel.


4. Slide the IPG strip into the ZOOM® Gel well.


5. Align the IPG strip properly in the ZOOM® Gel well using a thin plastic ruler or a weighing spatula. Avoid introducing any air bubbles while sliding the strip.


6. Pour ~ 400 µl of 0.5% agarose solution into the ZOOM® Gel well containing the IPG strip. Take care that the agarose solution does not overflow into the molecular weight marker well.


7. Assemble the gel cassette/Buffer Core sandwich as described in the XCell SureLock™ Mini-Cell manual. If you are using only one gel, use the Buffer Dam to replace the second gel cassette. Note: Do not use the ZOOM® IPGRunner™ Core for electrophoresis of the second dimension gel. You must use the Buffer Core supplied with the XCell SureLock™ Mini-Cell.


8. Fill the Lower Buffer Chamber and Upper Buffer Chamber with the appropriate running buffer.


9. Load molecular weight standards in the marker well.


10. 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].


11. Electrophorese at 200 V for 40 minutes for NuPAGE® Novex Bis-Tris ZOOM® Gel or at 125 V for 90 minutes for Novex® Tris-Glycine ZOOM® Gel.


12. At the end of electrophoresis, turn off the power and disassemble the gel cassette/Buffer Core sandwich assembly as described in the XCell SureLock™ Mini-Cell manual.


13. Proceed to staining the second dimension gel using any method of choice.

 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, and
• Secondary structure of the protein

The same 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 in the NuPAGE® Gels listed here 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 (collagens, histones, or highly hydrophobic membrane proteins) and in peptides, where the effect of local secondary structure 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 including the NuPAGE® buffer system. 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 the NuPAGE® Buffer System. You may generate calibration curves in your lab with any other manufacturer’s standards.

 
Using incorrect Buffers with NuPAGE^® Bis-Tris Gels

See the table below for the outcome of your results if you accidentally used an incorrect buffer instead of the NuPAGE^® MOPS/MES SDS Running Buffer and NuPAGE^® LDS Sample Buffer on the NuPAGE^® Bis-Tris Gels.

Using incorrect Buffers with NuPAGE^® Tris-Acetate Gels

NuPAGE® MOPS SDS Running Buffer

The NuPAGE® MOPS SDS Running Buffer (20X) is available from Thermo Fisher Scientific.
 
50 mM MOPS
50 mM Tris base
0.1% SDS
1 mM EDTA
pH 7.7

  1.   To prepare 500 ml of 20 X NuPAGE® MOPS SDS Running Buffer, dissolve the following reagents to 400 ml ultrapure water:

MOPS                                              104.6 g
Tris Base                                          60.6 g
SDS                                                     10 g
EDTA                                                  3.0 g

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

  3.   Store at +4° C. The buffer is stable for 6 months when stored at +4° C.

  4.   For electrophoresis, dilute this buffer to 1X with water. The pH of the 1X solution is 7.7. Do not use acid or base to adjust the pH.

NuPAGE® MES SDS Running Buffer

The NuPAGE® MES SDS Running Buffer (20X) is available from Thermo Fisher Scientific.
 
50 mM MES
50 mM Tris base
0.1% SDS
1 mM EDTA
pH 7.3

  1.   To prepare 500 ml of 20 X NuPAGE® MES SDS Running Buffer, dissolve the following reagents to 400 ml ultrapure water:

MES                                                     97.6 g
Tris Base                                             60.6 g
SDS                                                        10 g
EDTA                                                     3.0 g

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

  3.   Store at +4° C. The buffer is stable for 6 months when stored at +4° C.

  4.   For electrophoresis, dilute this buffer to 1X with water. The pH of the 1X solution is 7.3. Do not use acid or base to adjust the pH.
 
NuPAGE® Tris-Acetate SDS Running Buffer

The NuPAGE® Tris-Acetate SDS Running Buffer (20X) is available from Thermo Fisher Scientific.
 
50 mM Tricine
50 mM Tris base
0.1% SDS
pH 8.24

  1.   To prepare 500 ml of 20 X NuPAGE® Tris-Acetate SDS Running Buffer, dissolve the following reagents to 400 ml ultrapure water:

Tricine                                                 89.5 g
Tris Base                                            60.6 g
SDS                                                       10 g

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

  3.   Store at +4° C. The buffer is stable for 6 months when stored at +4° C.

  4.   For electrophoresis, dilute this buffer to 1X with water. The pH of the 1X solution is 8.24. Do not use acid or base to adjust the pH.

NuPAGE® Transfer Buffer

The NuPAGE® Transfer Buffer (20X) is available from Thermo Fisher Scientific.
 
25 mM Bicine
25 mM Bis-Tris (free base)
1 mM EDTA
pH 7.2

  1.   To prepare 125 ml of 20 X NuPAGE® Transfer Buffer, dissolve the following reagents to 100 ml ultrapure water:

Bicine                                                 10.2 g
Bis-Tris (free base)                           13.1 g
EDTA                                                  0.75 g

  2.   Mix well and adjust the volume to 125 ml with ultrapure water.

  3.   Store at +4° C. The buffer is stable for 6 months when stored at +4° C.

  4.   For western transfer, dilute this buffer to 1X with water. The pH of the 1X solution is 7.2. Do not use acid or base to adjust the pH.

NuPAGE® LDS Sample Buffer

The NuPAGE® LDS Sample Buffer (4X) is available from Thermo Fisher Scientific.
 
106 mM Tris HCl
141 mM Tris base
2% LDS
10% Glycerol
0.51 mM EDTA
0.22 mM SERVA® Blue G250
0.175 mM Phenol Red
pH 8.5

  1.   To prepare 10 ml of 4 X NuPAGE® LDS Sample Buffer, dissolve the following reagents to 8 ml ultrapure water:

Tris HCl                                                           0.666 g
Tris Base                                                        0.682 g
LDS                                                                 0.800 g
EDTA                                                               0.006 g
Glycerol                                                                 4 g
SERVA® Blue G250 (1% solution)                0.75 ml
Phenol Red (1% solution)                               0.25 ml

  2.   Mix well and 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.

  4.   For electrophoresis, prepare your samples in this buffer as described. The pH of the 1X solution is 8.5. Do not use acid or base to adjust the pH.

Tris-Glycine Native Sample Buffer

The Tris-Glycine Native Sample Buffer (2X) 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 2 X Tris-Glycine Native Sample Buffer, mix the following reagents:

4 M Tris HCl                                       4 ml
10% Glycerol                                     2 ml
0.1% Bromophenol Blue                 0.5 ml
Deionized Water                             3.5 ml

  2.   Mix well and adjust the pH of the solution is 8.6.

  3.   Store at +4° C. The buffer is stable for 6 months when stored at +4° C.

  4.   Use this buffer to prepare samples for non-denaturing NuPAGE® Tris-Acetate gel electrophoresis.

Tris-Glycine Native Running Buffer

The Tris-Glycine Native Running Buffer (10X) 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 in 900 ml deionized 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.

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. Moos, M Jr., Nguyen, N. Y., Liu, T.Y. (1988) Reproducible High Yield Sequencing of Proteins Electrophoretically Separated and Transferred to an Inert Support. J. Biol. Chem. 263, 6005-6008.


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