Introduction

The NuPAGE® Bis-Tris Electrophoresis System is a revolutionary neutral pH, discontinuous SDS-PAGE, pre-cast polyacrylamide mini-gel system. The neutral pH 7.0 environment during electrophoresis results in maximum stability of both proteins and gel matrix, providing better band resolution than other gel systems.
 
The Laemmli system is the most widely used SDS-PAGE method for separating a broad range of proteins (Laemmli, 1970). The highly alkaline operating pH of the Laemmli system may cause band distortion, loss of resolution, or artifact bands. The major causes of poor band resolution with the Laemmli system are:

  • 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).

Advantages of the NuPAGE® Electrophoresis System

The operating neutral pH of the NuPAGE® Gels and buffers provide following advantages over the Laemmli system:

  • 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
 

TOP

Ordering Information

Description of the NuPAGE Electrophoresis® System

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 Migration Chart on our Web site at www.invitrogen.com 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).
  

 
NuPAGE® Novex Bis-Tris Gels
 
NuPAGE® Novex Tris-Acetate Gels
Separating Gel Acrylamide Concentration
10%
3-8%
12%
7%
4-12%
 
Stacking Gel Acrylamide Concentration
4%
3.2%
Gel Thickness
1.0 mm
1.0 mm
1.5 mm
1.5 mm
Well Format
                1, 9, 10, 12, 15, 17, 2D, and IPG well
                             10, 12, 15, and 2D well


Formulation

The formulation for the NuPAGE® Gels is listed below:

NuPAGE® Novex Bis-Tris Gels
NuPAGE® Novex Tris-Acetate Gels
Bis-Tris-HCl buffer (pH 6.4)
Acrylamide
Bis-acrylamide
Ammonium persulfate (APS)
Ultrapure water
The separating gel operates at pH 7.0.
Tris base
Acetic acid
Acrylamide
Bis-acrylamide
TEMED
Ammonium persulfate (APS)
Ultrapure water
The separating gel operates at pH 8.1


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.

Well Types
Maximum Load Volume
Maximum Protein Load Per Band by Detection Method
  
Coomassie® Staining
Silver Staining
Immunoblotting
1.0 mm
700 µl
12 µg/band
Scale your sample load for the sensitivity of your silver staining kit.
For use with the SilverQuest or SilverXpress ® Silver Staining Kits, we recommend a protein load of 1 ng/band.
Scale your sample load according to the sensitivity of your detection method.
1.0 mm
                    1.5 mm
400 µl
600 µl
12 µg/band
  
1.0 mm
7 cm IPG Strip
N/A
  
1.0 mm
28 µl
0.5 µg/band
  
1.0 mm
                    1.5 mm
25 µl
37 µl
0.5 µg/band
  
1.0 mm
20 µl
0.5 µg/band
  
1.0 mm
                      1.5 mm
15 µl
25 µl
0.5 µg/band
  
1.0 mm
15 µl
0.5 µg/band
  


TOP

Preparing Sample

Introduction

General information on the sample buffer and reducing agent is provided below. For sample preparation protocols.
 
NuPAGE® LDS Sample Buffer

Use the NuPAGE® LDS Sample Buffer (4X) for preparing samples for denaturing gel electrophoresis with the NuPAGE® Gels.  For native gel electrophoresis with NuPAGE® Tris-Acetate Gels, use the Novex® Tris-Glycine Native Sample Buffer (2X).  For optimal sample preparation in all SDS-PAGE protocols, including the NuPAGE® system, denature and reduce the protein disulfide bonds under slightly alkaline pH conditions. Since the pH of the NuPAGE® LDS Sample Buffer is 8.4, sample reduction at this pH allows for maximal activity of the reducing agent.
 
The NuPAGE® LDS Sample Buffer is a 4X concentrated solution containing twice as much LDS as the 2X concentration of Novex® Tris-Glycine SDS or Tricine SDS Sample Buffer. This makes the NuPAGE® LDS Sample Buffer more viscous and difficult to pipet as compared to the Novex® Tris-Glycine or Tricine Buffers. The presence of more glycerol also increases the viscosity of the NuPAGE® LDS Sample Buffer. By bringing the NuPAGE® LDS Sample Buffer to room temperature (25°C), the buffer is more manageable
 
Tracking Dye

The NuPAGE® LDS Sample Buffer contains Coomassie G250 and Phenol Red as tracking dyes instead of bromophenol blue. Coomassie G250 gives a sharp dye front with both MES and MOPS SDS Running Buffers and migrates much closer to the moving ion front than bromophenol blue. This ensures that small peptides do not run off the gel. Bromophenol blue runs more slowly than some peptides with the MES SDS Running Buffer. The concentration of the tracking dye (Coomassie G250) is increased in the NuPAGE® LDS Sample Buffer to enhance viewing of the dye front.
 
Reducing Agent

The NuPAGE® Reducing Agent contains 500 mM dithiothreitol (DTT) at a 10X concentration and is available in a ready-to-use, stabilized liquid form. Use the NuPAGE® Reducing Agent to prepare samples for reducing gel electrophoresis. b-mercaptoethanol is compatible with the NuPAGE® system and can be used with the NuPAGE® gels at a final concentration of 2.5%. Choice of the reducing agent is a matter of preference and either DTT or b-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.
 
Do not use the NuPAGE® Antioxidant as a sample reducing agent. The antioxidant is not efficient in reducing the disulfide bonds. This will result in partially reduced bands with substantial background smearing in the lane.
The antioxidant maintains the sample proteins that have been previously reduced with a reducing agent in a reduced state and prevents the proteins from reoxidizing during 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, follow these guidelines:

  • 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.
  • If you are running reduced and non-reduced samples on the same gel, omit the antioxidant. The antioxidant will have a deleterious effect on the non-reduced samples. The bands will be sharper on NuPAGE® Gels relative to other gel systems, even without the use of the antioxidant.
 
Materials Supplied by the User

You will need the following items:

  • Protein sample
  • Deionized water

For denaturing electrophoresis

  • NuPAGE® LDS Sample Buffer (see recipe)
  • NuPAGE® Reducing Agent

For non-denaturing electrophoresis

  • Novex® Tris-Glycine Native Sample Buffer

Preparing Samples for Denaturing NuPAGE® Gel Electrophoresis

Instructions are provided below to prepare reduced or non-reduced samples for denaturing gel electrophoresis using the NuPAGE® Novex Bis-Tris or Tris-Acetate Gels.  The NuPAGE® LDS Sample Buffer and NuPAGE® Reducing Agent are available from Invitrogen.
 
For reduced sample, add the reducing agent immediately prior to electrophoresis to obtain the best results.

Reagent Reduced Sample Non-reduced Sample
Sample x µl x µl
NuPAGE® LDS Sample Buffer (4X) 2.5 µl2.5 µl
NuPAGE® Reducing Agent (10X) 1 µl --
Deionized Water to 6.5 µl to 7.5 µl
Total Volume 10 µl 10 µl

 
Preparing Samples for Non-Denaturing NuPAGE® Gel Electrophoresis

Instructions are provided below to prepare samples for non-denaturing (native) gel electrophoresis using the NuPAGE® Novex Tris-Acetate Gels. The Novex® Tris-Glycine Native Sample Buffer is available from Invitrogen.
 
Reagent Volume
Sample x µl
Novex® Tris-Glycine Native Sample Buffer (2X) 5 µl
Deionized Water to 5 µl
Total Volume 10 µl

Heating Samples
Heat the sample for denaturing electrophoresis (reduced or non-reduced) at 70°C for 10 minutes for optimal results.  Do not heat samples for non-denaturing (native) electrophoresis.

TOP

Preparing Running Buffer

Introduction

General information on the running buffer and antioxidant is provided below. Instructions for preparing running buffers for denaturing and non-denaturing electrophoresis are provided.
 
NuPAGE® SDS Running Buffer

Three types of NuPAGE® Running Buffers are used for denaturing gel electrophoresis of NuPAGE® Gels.

  • NuPAGE® MES SDS Running Buffer is used with NuPAGE® Novex Bis-Tris Gels to resolve small molecular weight proteins
  • NuPAGE® MOPS SDS Running Buffer is used with NuPAGE® Novex Bis-Tris Gels to resolve mid-size proteins
  • NuPAGE® Tris-Acetate SDS Running Buffer is used with NuPAGE® NovexTris-Acetate Gels to resolve high molecular weight proteins

The NuPAGE® MES SDS Running Buffer and NuPAGE® MOPS SDS Running Buffers have different pKa’s, resulting in MES being a faster running buffer than MOPS. The difference in ion migration affects the stacking and the separation ranges of proteins with these buffers.
 
For native gel electrophoresis with NuPAGE® NovexTris-Acetate Gels, use the Novex® Tris-Glycine Native Running Buffer.
 
NuPAGE® Antioxidant

The reducing agents, DTT and ß-mercaptoethanol, do not co-migrate through the gel with the sample in a neutral pH environment of the NuPAGE® Gels. Instead, the reducing agent tends to remain at the top of the gel and not migrate fully throughout the gel. Disulfide bonds are less reactive at neutral pH and are less likely to reoxidize than in a higher pH system. However, in the absence of an antioxidant some reoxidization may occur during the electrophoresis, resulting in slightly diffuse bands.
 
The NuPAGE® Antioxidant (a proprietary reagent) is added to the running buffer in the upper (cathode) buffer chamber only when performing electrophoresis under reducing conditions. The NuPAGE® Antioxidant migrates with the proteins during electrophoresis preventing the proteins from reoxidizing and maintaining the proteins in a reduced state. The NuPAGE® Antioxidant also protects sensitive amino acids such as methionine and tryptophan from oxidizing.
We also recommend using the NuPAGE® Antioxidant with reduced samples that have been alkylated, for optimal results.
 
The NuPAGE® Antioxidant is NOT compatible with gel systems other than the NuPAGE® system as the antioxidant is not efficient at higher pHs of other gel systems.
 
Materials Supplied by the User

You will need the following items:

  • Deionized water

For denaturing electrophoresis

  • NuPAGE® MES or MOPS SDS Running Buffer (see recipes)
  • NuPAGE® Tris Acetate Running Buffer (see recipes)
  • NuPAGE® Antioxidant

For non-denaturing electrophoresis

  • Novex® Tris-Glycine Native Running Buffer  

Preparing Buffer for Denaturing Electrophoresis

NuPAGE® SDS Running Buffer (20X) is available from Invitrogen.
 
Reducing Conditions

  1. Prepare 1000 ml of 1X NuPAGE® SDS Running Buffer using NuPAGE® SDS Running Buffer (20X) as follows:

  2. NuPAGE® SDS Running Buffer (20X, MES, MOPS, or Tris-Acetate)               50 ml
    Deionized Water                                                                                               950 ml    
    Total Volume                                                                                                    1000 ml


  3. Mix thoroughly and set aside 800 ml of the 1X NuPAGE® SDS Running Buffer for use in the Lower (Outer) Buffer Chamber of the XCell SureLock™ Mini-Cell.

  4. Immediately, prior to electrophoresis, add 500 µl of NuPAGE® Antioxidant to 200 ml of 1X NuPAGE® SDS Running Buffer from Step 1 for use in the Upper (Inner) Buffer Chamber of the XCell SureLock™ Mini-Cell. Mix thoroughly.


Non-Reducing Conditions

  1. Prepare 1000 ml of 1X NuPAGE® SDS Running Buffer using NuPAGE® SDS Running Buffer (20X) as follows:

  2. NuPAGE® SDS Running Buffer (20X, MES or MOPS)                  50 ml
    Deionized Water                                                                           950 ml
    Total Volume                                                                               1000 ml


  3. Mix thoroughly. Fill the Upper and Lower Buffer Chamber of the XCell SureLock™ Mini-Cell with this Running Buffer.


See recipe of the NuPAGE® SDS Running Buffers, if you are preparing the running buffers.

Preparing Buffer for Non-Denaturing Electrophoresis

Novex® Tris-Glycine Native Running Buffer (10X) is available from Invitrogen. Prepare 1000 ml of 1X Native Running Buffer using the Novex® Tris-Glycine Native Running Buffer (10X) as follows:
 

  1. Novex® Tris-Glycine Native Running Buffer (10X)                 100 ml
  2. Deionized Water                                                                               900 ml
    Total Volume                                                                                   1000 mlMix thoroughly.

  3. Mix thoroughly and use 800 ml of this Running Buffer in the Lower and Upper Buffer Chambers of the XCell SureLock™ Mini-Cell.


See recipe of the Novex® Tris-Glycine Native Running Buffer, if you are preparing the running buffer.
 
Note

  • If you forget to add the antioxidant to the upper buffer chamber, some bands may be slightly fuzzier and more diffuse due to reoxidation of some proteins during electrophoresis.
  • We recommend preparing the running buffer for the upper chamber with the antioxidant within half an hour of planned use. If the antioxidant is added to the running buffer too long before use, gels may exhibit signs of reoxidation (slightly fuzzier bands).  
  • If you have added 0.5 ml of antioxidant to the total amount of buffer (for upper and lower buffer chamber) by accident, the required concentration of antioxidant will be lower and the antioxidant will not be effective. If you wish to add antioxidant to the total amount of buffer (for upper and lower buffer chamber), add 2.5 ml of antioxidant to obtain the expected results. However, this is not recommended as high current will be generated and the antioxidant is wasted.
  • If you have switched the antioxidant and the reducing agent (used the antioxidant in the sample buffer and the reducing agent in the running buffer) by accident, the reducing agent (DTT) will not migrate into the gel and the antioxidant will not effectively reduce the samples, resulting in decreased staining sensitivity.


TOP

Protocol - Electrophoresis of NuPAGE® Gels

 

Introduction

Instructions are provided below for electrophoresis of the NuPAGE® Gels using the XCell SureLock™ Mini-Cell. For more information on the XCell SureLock™ Mini-Cell, refer to the manual (IM-9003). This manual is available on our Web site at www.invitrogen.com or contact Technical Service.
If you are using any other electrophoresis mini-cell, follow the manufacturer’s recommendations.
 
To ensure success with the NuPAGE®  Electrophoresis System, remember the important points listed below:

  • Under NO circumstances should Tris-Glycine SDS buffers be used with NuPAGE® Gels for any denaturing gel electrophoresis (see troubleshooting for the outcome of your results using incorrect buffers)
  • Use ONLY NuPAGE® SDS buffers
  • DO NOT BOIL samples. Heat samples at 70° C for 10 minutes. Inner and Outer Buffer Chambers MUST be filled with the recommended amount of running buffer to prevent excessive heating.
 
Procedure using XCell SureLock™ Mini-Cell

Wear protective gloves and safety glasses when handling gels.
XCell SureLock™ Mini-Cell require 200 ml for the Upper Buffer Chamber and 600 ml for the Lower Buffer Chamber.

  1. Remove the NuPAGE® 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 1X NuPAGE® SDS Running Buffer. Invert the gel and shake 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 (see recipe for running buffer preparation). The buffer level must exceed the level of the wells. Note: If you are running reduced samples, remember to fill the Upper Buffer Chamber with 200 ml of running buffer containing the NuPAGE® Antioxidant.

  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 (outer) Buffer Chamber with 600 ml of the appropriate 1X running buffer.
 
Electrophoresis Conditions

Run your gels according to the following protocol:

Gel Type
Voltage
Expected Current*
Run Time
NuPAGE® Novex Bis-Tris Gels with MES SDS Running Buffer
200 V constant†
Start: 110-125 mA/gel
End: 70-80 mA/gel
35 minutes
NuPAGE® Novex Bis-Tris Gels with MOPS SDS Running Buffer
200 V constant†
Start: 100-115 mA/gel
End: 60-70 mA/gel
50 minutes
NuPAGE® Novex Tris-Acetate Gels
150 V constant
Start: 40-55 mA/gel
End: 25-40 mA/gel
1 hour
NuPAGE® Novex Tris-Acetate Native Gels
150 V constant
Start: 18 mA/gel
End: 7 mA/gel
~2 hours
Run times may vary

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 to the Western Transfer Protocol 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.

TOP

Using ZOOM® Gels

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 web site at www.invitrogen.com 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.

TOP

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, 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.

Mark 12 Unstained Standard
NuPAGE® (4-12%)
Bis-Tris/MES
NuPAGE® (4-12%)
Bis-Tris/MOPS
NuPAGE® (3-8%)
Tris-Acetate
Myosin
200 kDa
200 kDa
200 kDa
ß-Galactosidase
116.3 kDa
116.3 kDa
116.3 kDa
Phosphorylase B
97.4 kDa
97.4 kDa
97.4 kDa
Bovine Serum Albumin
66.3 kDa
66.3 kDa
66.3 kDa
Glutamic Dehydrogenase
55.4 kDa
55.4 kDa
55.4 kDa
Lactate Dehydrogenase
36.5 kDa
36.5 kDa
36.5 kDa
Carbonic Anhydrase
31 kDa
31 kDa
31 kDa
Trypsin Inhibitor
21.5 kDa
21.5 kDa
N/A
Lysozyme
14.4 kDa
14.4 kDa
N/A
Aprotinin
6 kDa
6 kDa
N/A
Insulin B Chain
3.5 kDa
N/A
N/A
Insulin A Chain
2.5 kDa
N/A
N/A

 

MultiMark® Multi-Colored Standard
NuPAGE® (4-12%)
Bis-Tris/MES
NuPAGE® (4-12%)
Bis-Tris/MOPS
NuPAGE® (3-8%)
Tris-Acetate
Myosin
185 kDa
188 kDa
209 kDa
Phosphorylase B
98 kDa
97 kDa
111 kDa
Glutamic Dehydrogenase
52 kDa
52 kDa
52 kDa
Carbonic Anhydrase
31 kDa
33 kDa
34 kDa
Myoglobin—Blue
19 kDa
21 kDa
N/A
Myoglobin—Red
17 kDa
19 kDa
N/A
Lysozyme
11 kDa
12 kDa
N/A
Aprotinin
6 kDa
N/A
N/A
Insulin
3 kDa
N/A
N/A

 

SeeBlue® Pre-Stained Standard
NuPAGE® (4-12%)
Bis-Tris/MES
NuPAGE® (4-12%)
Bis-Tris/MOPS
NuPAGE® (3-8%)
Tris-Acetate
Myosin
188 kDa
191 kDa
210 kDa
BSA
62 kDa
64 kDa
71 kDa
Glutamic Dehydrogenase
49 kDa
51 kDa
55 kDa
Alcohol Dehydrogenase
38 kDa
39 kDa
41 kDa
Carbonic Anhydrase
28 kDa
28 kDa
N/A
Myoglobin
18 kDa
19 kDa
N/A
Lysozyme
14 kDa
14 kDa
N/A
Aprotinin
6 kDa
N/A
N/A
Insulin
3 kDa
N/A
N/A

 

SeeBlue® Plus2 Pre-Stained Standard
NuPAGE® (4-12%)
Bis-Tris/MES
NuPAGE® (4-12%)
Bis-Tris/MOPS
NuPAGE® (3-8%)
Tris-Acetate
Myosin
188 kDa
191 kDa
210 kDa
Phosphorylase B
98 kDa
97 kDa
111 kDa
BSA
62 kDa
64 kDa
71 kDa
Glutamic Dehydrogenase
49 kDa
51 kDa
55 kDa
Alcohol Dehydrogenase
38 kDa
39 kDa
41 kDa
Carbonic Anhydrase
28 kDa
28 kDa
N/A
Myoglobin
17 kDa
19 kDa
 

 

TOP

Troubleshooting

Problem
Cause
Solution
Run taking longer time
Running buffer too dilute
Make fresh running buffer as described and do not adjust 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.
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.
Dumbbell shaped bands after electrophoresis
Loading a large volume of sample causes incomplete stacking of the entire sample. This effect is more intensified for larger proteins
Load the appropriate volume of sample per well as described. If your sample is too dilute, concentrate the sample using ultrafiltration.

 

 
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.

 

If you used the…
Instead of the….
Then….
NuPAGE® MES SDS Running Buffer
NuPAGE® MOPS SDS Running Buffer
  • the run time of the gel is decreased by ~10-15 minutes.
  • there is decreased separation and resolution for proteins > 36 kDa.
NuPAGE® MOPS SDS Running Buffer
NuPAGE® MES SDS Running Buffer
  • the run time of the gel is increased by ~10-15 minutes.
  • the lower molecular weight proteins (<14 kDa), which are normally well resolved, are not resolved while the high molecular weight proteins are resolved more than normal.
Novex® Tris-Glycine SDS Sample Buffer
NuPAGE® LDS Sample Buffer
some bands are not very sharp and there is increased protein fragmentation.
Novex® Tricine SDS Sample Buffer
NuPAGE® LDS Sample Buffer
the band sharpness is not affected, but the lanes will be slightly wider due to the increased amount of SDS and buffer salts from the Tricine Sample Buffer.
Novex® Tris-Glycine SDS Running Buffer and the Novex® Tris-Glycine SDS Sample BufferNuPAGE® MOPS or MES SDS Running Buffer and the NuPAGE® LDS Sample Buffer
  • the sensitivity of the staining for high molecular weight proteins is decreased.
  • the bands are more compressed at the bottom of the gel, regardless of the gel percentage and the bands have a cupped appearance at the bottom of the band.
  • the gel will have an extremely long run time of 3-4 hours due to the low migration of the glycine ions at neutral pH.
Novex® Tricine SDS Running Buffer and the Tricine SDS Sample Buffer
NuPAGE® MOPS or MES SDS Running Buffer and the NuPAGE® LDS Sample Buffer
  • the run time of the gel is increased by 1-2 hours due to the slow migration of the tricine ions at neutral pH.
  • there may be background streaking in the lanes.

Using incorrect Buffers with NuPAGE® Tris-Acetate Gels

Refer to the table below for the outcome of your results if you accidentally used a incorrect buffer system instead of the NuPAGE® Tris-Acetate SDS Running Buffer and NuPAGE® LDS Sample Buffer on the NuPAGE® Tris-Acetate Gels.
 
Sample Buffer
Running Buffer
Antioxidant
Results
Novex® Tris-Glycine SDS
NuPAGE® Tris-Acetate SDS
Yes
Fuzzy, smeary bands.
Novex® Tricine SDS
NuPAGE® Tris-Acetate SDS
Yes
Bands are not very sharp.
NuPAGE® LDS
NuPAGE® MES SDS or NuPAGE® MOPS SDS
Yes
Bands are diffuse and have a “U” shape. More low molecular weight proteins are visible.
NuPAGE® LDS
Novex® Tris-Glycine SDS
No
The run time is twice as long as the Tris-Acetate Buffer system. The band resolution is poor.
NuPAGE® LDS
Novex® Tricine SDS
No
The run time is 10-15 minutes faster than the Tris-Acetate Buffer system. Reduced protein bands are diffuse while non-reduced large molecular weight protein bands are smeary.
Novex® Tris-Glycine SDS
Novex® Tris-Glycine SDS
No
The run time is much longer than the Tris-Acetate Buffer system and the bands are very faint with a streaking background. Fewer low molecular weight bands are resolved.
Novex® Tricine SDS
Novex® Tricine SDS
No
The run time is 10-15 minutes faster than the Tris-Acetate Buffer system and reduced protein bands are not very sharp. The overall performance is acceptable.


TOP

 

Recipes

NuPAGE® MOPS SDS Running Buffer

The NuPAGE® MOPS SDS Running Buffer (20X) is available from Invitrogen
 
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 Invitrogen
 
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 Invitrogen
 
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 Invitrogen
 
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 Invitrogen
 
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 Invitrogen

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 Invitrogen

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.



TOP

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. 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.


TOP
LT057