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NuPAGE® Electrophoresis System Components
The NuPAGE® Electrophoresis System consists of:
NuPAGE® Bis-Tris Discontinuous Buffer System
The NuPAGE® Bis-Tris discontinuous buffer system involves three ions:
NuPAGE® Tris-Acetate Discontinuous Buffer System
The NuPAGE® Tris-Acetate discontinuous buffer system involves three ions:
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).
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:
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
|
Reagent | Reduced Sample | Non-reduced Sample |
---|---|---|
Sample | x µl | x µl |
NuPAGE® LDS Sample Buffer (4X) | 2.5 µl | 2.5 µl |
NuPAGE® Reducing Agent (10X) | 1 µl | -- |
Deionized Water | to 6.5 µl | to 7.5 µl |
Total Volume | 10 µl | 10 µl |
Reagent | Volume |
---|---|
Sample | x µl |
Novex® Tris-Glycine Native Sample Buffer (2X) | 5 µl |
Deionized Water | to 5 µl |
Total Volume | 10 µl |
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
|
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
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:
Equilibrating the IPG Strip
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.
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:
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
|
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
|
|
Streaking of proteins
|
|
|
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
|
|
NuPAGE
® MOPS SDS Running Buffer
|
NuPAGE
® MES SDS Running Buffer
|
|
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 Buffer | NuPAGE® MOPS or MES SDS Running Buffer and the NuPAGE® LDS Sample Buffer |
|
Novex
® Tricine SDS Running Buffer and the Tricine SDS Sample Buffer
|
NuPAGE
® MOPS or MES SDS Running Buffer and the NuPAGE
® LDS Sample Buffer
|
|
Using incorrect Buffers with 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.
|
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.