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NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels
NuPAGE Bis-Tris Gels and Bolt Bis-Tris Plus Gels are high-performance precast polyacrylamide gels developed to provide optimal separation of a wide range of protein sizes under denaturing conditions. The neutral pH Bis-Tris chemistry delivers consistent gel performance and minimizes protein modifications that can lead to protein degradation. As a result, NuPAGE and Bolt gels are a good choice for downstream transfer and sensitive western blot analysis and any other technique where protein integrity is crucial. The unique WedgeWell design of the Bolt Bis-Tris Plus gel allows loading of up to 2X more sample volume than conventional precast gels. NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels are also stable at room temperature, which helps save refrigerator space.
NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gel specifications
| NuPAGE Bis-Tris | Bolt Bis-Tris Plus | |
|---|---|---|
| Select when | Need flexibility in well and gel format, or when you need a higher-throughput midi gel for a larger number of samples | Working with dilute samples or need to load larger sample volumes, or when faster run times are needed |
| Available gel sizes | Mini: 8 cm x 8 cm (1.0 or 1.5 mm thick) Midi: 8 cm x 13 cm (1.0 mm thick) | Mini: 8 cm x 8 cm (1.0 mm thick) |
| Storage conditions | 4–25°C | |
| Shelf life | 16 months | 16 months |
| High lot-to-lot consistency | Coefficient of variation (CV) of only 2% for Rf values (migration) | Coefficient of variation (CV) of only 2% for Rf values (migration) |
| Recommended sample buffer | NuPAGE LDS Sample Buffer | Bolt LDS sample buffer |
| Recommended running buffers | NuPAGE MES SDS or NuPAGE MOPS SDS Running Buffer | Bolt MES SDS or Bolt MOPS SDS running buffer |
| Run time | 35 min (MES buffer) 50 min (MOPS Buffer) | 20 min (MES buffer) 35 min (MOPS buffer) |
| Recommended transfer buffer | NuPAGE Transfer Buffer | Bolt Transfer Buffer |
| Gel chemistry | Bis-Tris | Bis-Tris Plus |
| Available polyacrylamide concentrations | 8%, 10%, 12%, 4–12% | |
| Separation range | 15 kDA to 260 kDa (MOPS buffer) 3.5 kDA to 160 kDa (MES buffer) | |
| For use with (equipment) mini gels | Mini Gel Tank or XCell SureLock Mini-Cell | |
| For use with (equipment) midi gels | SureLock Tandem Midi Gel Tank, Invitrogen XCell4 SureLock Midi-Cell or Bio-Rad Criterion (with adapters only) | Not available in midi size |
| Mode of separation | Molecular weight | |
| Application | SDS-PAGE | |
| Available Wells configurations* | Mini: 1, 9, 10, 12, 15, 17, 2D-well, IPG well Midi: 12+2, 20, 26 wells | WedgeWell format: 10, 12, 15 17 well (load up to 2X sample volume per well) |
*Not all percentages are available in every well type
Invitrogen Bis-Tris Gel Highlights
Sharp, straight bands
Neutral-pH buffers in NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels deliver sharp straight bands. During separation, the gels operate close to pH 7. In the Laemmli system (Tris-glycine), the gel is run at basic pH (pH ~9.5). At high pH the residual unpolymerized acrylamide can react with cysteine and lysine residues of the proteins being separated. This may affect downstream analysis such as western blot transfer. At the neutral pH of NuPAGE Bis-Tris and Bolt Bis-Tris Gels, these reactions take place several orders of magnitude more slowly than at the basic pH of Tris-glycine gels, resulting in sharper bands and better resolution.
Protein separation comparing NuPAGE Bis-Tris Gel and traditional tris-glycine gel. The samples listed below were run on (A) a NuPAGE 4–12% Bis-Tris Gel in Invitrogen NuPAGE MES SDS Running Buffer or (B) another manufacturer’s 4–20% tris-glycine gel. Note the high resolution of the sample bands in the NuPAGE protein gel. The poorly resolved “fuzzy” bands seen in the other manufacturer’s gel are a result of reoxidation of some disulfide bonds within the sample, leading to slight changes in migration rates. Lanes 1, 6, 7, 10: Invitrogen Mark12 Unstained Standard; lane 2: high protein load (4 proteins, 6 µg/protein); lane 3: Broad Range Standard; lanes 4, 5: E. coli extract; lane 8: E-PAGE SeeBlue Pre-stained Standard; lane 9: Invitrogen MultiMark Multi-Colored Protein Standard.
Higher-throughput electrophoresis using NuPAGE Bis-Tris Midi Gels
Publication-quality protein electrophoresis data is easily generated using the higher-throughput NuPAGE Bis-Tris mide gels and the SureLock Tandem Midi Gel Tank. The gel produces crisp bands and straight protein lanes.
NuPAGE Bis-Tris 4–12% gradient midi gel result
Publication-quality protein electrophoresis gel results using NuPAGE Bis-Tris 4-12% gradient midi gels and SureLock Tandem Midi Gel Tank. Samples were loaded as follows: Lanes 1, 20: 5 µL PageRuler Broad Range Unstained Protein Ladder; lanes 2–7: 10 µg, 8 µg, 6 µg, 4 µg, 2 µg, 1 µg HeLa lysate, respectively; lanes 8, 13: 5 µL Novex Mark12 Unstained; lanes 9–12: 240 ng, 180 ng, 120 ng, 60 ng of protein mix containing β-galactosidase, lactate dehydrogenase, and lysozyme; lanes 14–19: 10 µg, 8 µg, 6 µg, 4 µg, 2 µg, 1 µg E. coli lysate, respectively. Electrophoresis was conducted with NuPAGE MOPS running buffer and the gel was stained with SimplyBlue Safe Stain.
Better sample integrity
Sample preparation is a crucial step in successful protein analysis. The pH of the traditional Laemmli-style sample buffer changes from 6.8 to 5.2 when heated at 100°C. This lower pH is known to induce aspartyl-prolyl (Asp-Pro) peptide bond cleavage, which leads to protein degradation. By maintaining a >7.0 pH environment, the Invitrogen NuPAGE LDS Sample Buffer and Bolt LDS Sample Buffer preserve protein integrity by minimizing this Asp-Pro cleavage. As seen below, the sample integrity is maintained throughout electrophoresis with the Invitrogen NuPAGE system; protein degradation is seen in samples prepared with Laemmli (Tris-glycine) sample buffer. The Invitrogen NuPAGE Antioxidant running buffer additive greatly minimizes protein oxidation during electrophoresis and keeps reduced protein bands sharp and clear.
High sample integrity. Integrity of samples is maintained throughout electrophoresis with the NuPAGE Bis-Tris Gel System (left), compared to samples prepared with Laemmli (tris-glycine) sample buffer (right).
Bolt Bis-Tris Plus Gel high capacity WedgeWell sample wells
The unique wedge-shaped well in every Bolt gel provides higher loading capacity, so you can load up to twice as much protein sample in every well.
| Well size | Recommended loading volume | Maximum loading volume | Maximum protein load |
|---|---|---|---|
| 10-well | 40 µL | 60 µL | 0.5 µg/band |
| 12-well | 30 µL | 45 µL | 0.4 µg/band |
| 15-well | 20 µL | 35 µL | 0.25 µg/band |
| 17-well | 15 µL | 30 µL | 0.2 µg/band |
Detect proteins even in dilute samples or measure expression of low-abundance proteins. With more room to load your sample, you no longer have to worry about sample spillover and cross-well contamination on your western blots. Gel loading is also easier with no need to use special gel-loading pipette tips.
High sample volume capacity of Bolt Bis-Tris Plus gels. (A) Increasing volumes (40–70μL) of a fluorescent protein standard were loaded in every other lane of an Invitrogen Bolt 4–12% Bis-Tris Plus 10-well Gel. (B) Increasing volumes (10–40 μL) of the same protein standard were loaded in every other lane of Bio-Rad's 4–20% 10-well gel. Sample spillover and cross-well contamination is observed as signals from the sample loaded in the adjacent wells.
Comparison of electrophoresis and western blotting of cell lysates using NuPAGE midi gels versus Bio-Rad
The amount of protein you can load into a protein gel well affects the ability to detect the protein following protein gel electrophoresis or western blotting; the more you can load, the easier it is to detect. The protein load capacities of Invitrogen NuPAGE Bis-Tris precast midi gels run in the SureLock Tandem Mid Gel Tank were compared against Bio-Rad Criterion midi gels run in a Bio-Rad Criterion Cell Midi Cell Tank using manufacturer instructions. Decreasing amounts of HEK 293 cell lysate prepared in RIPA lysis buffer (48–0.5 µg total protein) were denatured in the respective manufacturer’s sample buffer and subjected to electrophoresis using manufacturer instructions. The table below lists the samples, protein mass, and %RIPA buffer loaded in each lane.
| Lane | +1 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | +1 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Sample | Sample buffer | iBright Protein Ladder | HEK293 lysate | iBright Protein Ladder | Sample buffer | |||||||||
| Load mass (µg) | - | - | 48 | 40 | 32 | 24 | 16 | 8 | 4 | 2 | 1 | 0.5 | - | - |
| Load vol. (µL) | 3 | 3 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 3 | 3 |
| % RIPA buffer | - | - | 40 | 33.3 | 26.7 | 20 | 13.3 | 6.7 | 3.3 | 1.7 | 0.8 | 0.4 | - | - |
Invitrogen NuPAGE Bis-Tris gels outperformed Bio-Rad gels at higher lysate loads with blots from Bio-Rad gels showing band loss and smearing at higher loads for all targets investigated.
Comparison of protein blotting and western detection of cell lysates
Western blots using NuPAGE 4-12% Bis-Tris midi gels generate sharper bands at greater protein and RIPA lysis buffer loads than Bio-Rad 4–20% TGX midi gels.A NuPAGE 4–12% Bis-Tris midi gel, 12+2 well, was loaded with decreasing total protein amount of HEK293 lysate, subjected to electrophoresis in a SureLock Tandem Midi Gel Tank and transferred onto a 0.45 µm PVDF membrane using the SureLock Tandem Blot Module and the Bio-Rad 4–20% TGX midi gel, 12+2 well, was subjected to electrophoresis in a Criterion Midi Cell Tank and transferred onto a 0.45 µm PVDF membrane using the Criterion Blotter. Both membranes were probed for three targets (vinculin, α-tubulin, and p23) using fluorescent immunodetection. Band loss and smearing can be seen at the higher loads for all targets on the Bio-Rad blot, while the blot for the NuPAGE Bis-Tris gel offers superior protein loading capacity above 24 µg and crisp, bright bands.
For immunodetection: membranes were blocked for 1 hour in 1X Blocker FL Fluorescent Blocking Buffer. For fluorescence: multiplex detection, the membranes were probed overnight with a mixture of primary antibodies diluted in blocking solution: Rabbit-anti Vinculin (1:15,000), Rat anti-α Tubulin (1:5,000)), and Mouse-anti p23 (1:5,000) followed by an incubation with secondary antibodies diluted at 1:5,000 in 1X Blocker FL: Donkey anti-Rabbit Alexa Fluor Plus 680, Donkey anti-Rat Alexa Fluor 488, and Donkey anti-Mouse Alexa Fluor Plus 800 for 1 hour. Membranes were imaged on an iBright Imaging System, with the same time and image adjustment settings for each blot.
NuPAGE Bis-Tris Gels and Bolt Bis-Tris Plus Gels
NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels are precast polyacrylamide gels designed to give optimal separation for a wide range of molecular weight proteins during gel electrophoresis under denaturing conditions. The Bis-Tris gel chemistry provides a neutral-pH environment that minimizes protein modifications, resulting in less protein degradation and sharper bands without typical gel "smiling". preserving protein integrity becomes particularly important when separating low-abundance proteins.
NuPAGE Bis-Tris gels are offered as mini and midi gels, and Bolt Bis-Tris Plus gels are offered as mini gels. Both NuPAGE Bis-Tris gels and Bolt Bis-Tris Plus gels are ideal for western blot analysis and applications such as sequencing and mass spectrometry where protein integrity is crucial and higher sensitivity is required. The unique WedgeWell design of the Bolt Bis-Tris Plus gel sample well allows loading of up to 2x more sample volume than other precast gels and the Bis-Tris Plus chemistry enables Bolt gels to be run in as little as 20 minutes.
Features of the NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels include:
- Better protein integrity—optimized sample preparation process preserves your proteins
- Wide ranges of molecular weight separation—selecting the right gel and running buffer helps to achieve the optimal separation of your proteins
- Faster run times—separation of your proteins can be attained in 20-35 minutes
- Longer shelf life—NuPAGE Bis-Tris and Bolt Bis-Tris Plus gels can be stored for 16 months at room temperature.
- High capacity wells of Bolt Bis-Tris Plus Gels—wedge-shaped wells makes sample loading easier (no specialized pipette tips required) and allow loads of up to two times higher sample volume
NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels are formulated for denaturing gel electrophoresis applications. For optimal sample preparation, we recommend using buffers containing SDS. The gels can be run using MES SDS running buffer to better resolve smaller proteins (1–200 kDa) and MOPS SDS running buffer to resolve medium- to large-size proteins (14–260 kDa).
Bis-Tris Gels use a Bis-Tris discontinuous buffer system involving three ions:
- Chloride (–) is from the gel buffer and serves as a leading ion due to its high affinity to the anode relative 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 in the running buffer. 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 lower pH gel buffer (pH 6.4) and the running buffer (pH 7.3–7.7) results in a significantly lower operating pH of 7 during electrophoresis.
Get started with Protein gels welcome packs
Protein gels welcome packs are a cost-effective way to start using Invitrogen precast protein gels for the first time. Protein gel welcome packs are available for each of the protein gel chemistries and percentages and come with mini or midi gels, buffers, ladders, and a Mini Gel Tank or SureLock Tandem Midi Gel Tank to provide you with great savings over the cost of the individual components.
Optimized performance of NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels
Unlike traditional Tris-glycine gels, NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels are Bis-Tris HCl buffered (pH 6.4) and have an operating pH of about 7.0. This neutral pH paired with a unique, gentle sample preparation protocol means your protein samples always reside in mild, nonacidic conditions, preserving the integrity of the proteins and minimizing protein modifications. This helps to ensure highly sensitive, accurate western blot results every time.
Greater sensitivity with Bolt Bis-Tris Plus gels. Total cell extracts from A431 cells were transferred to NC and PVDF membranes from a 4–12% Bolt Bis-Tris Plus gel, and 4–20% Tris-glycine precast gel using the iBlot 2 Gel Transfer Device. The cells were treated with 100 ng/mL of human epidermal growth factor (hEGF) to up-regulate expression of the phospho-EGF receptor. The protein loads of the cell extracts ranged from 20 μg to 1.2 μg of extract. The blots were processed on the iBind Western System using a 1:200 dilution of Phospho-EGF Receptor (Tyr1068) (1H12) Mouse mAb (Cell Signaling Technology) and a 1:2,000 dilution of anti-mouse HRP secondary antibody.
Band integrity
NuPAGE and Bolt gels preserve protein integrity with neutral-pH gel chemistry. This formulation minimizes protein modifications during electrophoresis.
A western blot of a Bolt Bis-Tris Plus gel shows clean, sharp protein signals corresponding to only full-length proteins, whereas a western blot of a Bio-Rad TGX gel shows multiple low molecular weight degradation products. Protein kinases implicated in cancer (IKKϐ, EPHB3, HCK, MAPK14, FLT1 and DDR2) were analyzed on a Bolt Bis-Tris Plus Gel and a Bio-Rad TGX tris-glycine gel. The purified kinases (50 ng each), along with MagicMark protein standard and purified recombinant GST protein, were loaded on a 10-well, 4–12% Bolt gel and a 10-well, 4–20% Bio-Rad TGX gel. The samples were separated and transferred to 0.45-μm PVDF membranes using the respective manufacturers’ protocols. Immunodetection was performed using an anti-GST antibody and WesternBreeze chemiluminescence detection.
Band resolution
NuPAGE and Bolt Bis-Tris Plus Gels have better resolving power than Bio-Rad’s gels. With 10% greater resolving distance, optimized gel chemistry, and gradient format, you can see more protein bands resolved on a Bolt gel. In experimental analysis, you can see two separate bands for the insulin B chain and insulin A chain, which have similar molecular weights. The protein bands on the Bolt gel appear straight and the signal intensities are even across the entire bands, whereas the protein bands on Bio-Rad’s gel are uneven and of poor quality.
Bolt Bis-Tris Gel band resolution—comparison of Bio-Rad's stained protein gels.(A) Bolt Bis-Tris gradient gel stained with Invitrogen SimplyBlue SafeStain. (B) Bio-Rad's tris-glycine gradient gel stained with competitor’s stain. (C) Enlargement of a section of lane 5 from the two gels shows the insulin B chain (3.5 kDa) and insulin A chain (2.5 kDa).
Use the protein gel selection guide to find the best mini or midi gel for your protein electrophoresis experiment.
Find the best Invitrogen mini or midi gel to replace your current precast gel from another supplier using the protein gel conversion tool.
Currently using Tris-glycine or Tricine gels? Use the NuPAGE Gel conversion guide below to find the appropriate NuPAGE gel and corresponding optimized buffer.
NuPAGE Gel conversion guide
| If you are using: | Then we recommend NuPAGE gel: | Buffer system |
|---|---|---|
| Tris-Glycine gel | ||
| 4% | NuPAGE 3-8% Tris-Acetate | Tris-Acetate |
| 6% | NuPAGE 3-8% Tris-Acetate | Tris-Acetate |
| 10% | NuPAGE 8% Bis-Tris | MOPS |
| NuPAGE 10% Bis-Tris | MOPS | |
| 12% | NuPAGE 10% Bis-Tris | MOPS |
| 14% | NuPAGE 12% Bis-Tris | MOPS |
| 16% | NuPAGE 12% Bis-Tris | MES |
| 18% | NuPAGE 12% Bis-Tris | MES |
| 4-12% | NuPAGE 3-8% Tris-Acetate | Tris-Acetate |
| NuPAGE 4-12% Bis-Tris | MOPS | |
| 4-20% | NuPAGE 4-12% Bis-Tris | MES |
| 8-16% | NuPAGE 4-12% Bis-Tris | MOPS |
| 10-20% | NuPAGE 12% Bis-Tris | MOPS |
| Tricine gel | ||
| 10% | NuPAGE 8% Bis-Tris | MES |
| NuPAGE 10% Bis-Tris | MES | |
| 16% | NuPAGE 4-12% Bis-Tris | MES |
| 10-20% | NuPAGE 4-12% Bis-Tris | MES |
Manuals
Handbooks
NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gel specifications
| NuPAGE Bis-Tris | Bolt Bis-Tris Plus | |
|---|---|---|
| Select when | Need flexibility in well and gel format, or when you need a higher-throughput midi gel for a larger number of samples | Working with dilute samples or need to load larger sample volumes, or when faster run times are needed |
| Available gel sizes | Mini: 8 cm x 8 cm (1.0 or 1.5 mm thick) Midi: 8 cm x 13 cm (1.0 mm thick) | Mini: 8 cm x 8 cm (1.0 mm thick) |
| Storage conditions | 4–25°C | |
| Shelf life | 16 months | 16 months |
| High lot-to-lot consistency | Coefficient of variation (CV) of only 2% for Rf values (migration) | Coefficient of variation (CV) of only 2% for Rf values (migration) |
| Recommended sample buffer | NuPAGE LDS Sample Buffer | Bolt LDS sample buffer |
| Recommended running buffers | NuPAGE MES SDS or NuPAGE MOPS SDS Running Buffer | Bolt MES SDS or Bolt MOPS SDS running buffer |
| Run time | 35 min (MES buffer) 50 min (MOPS Buffer) | 20 min (MES buffer) 35 min (MOPS buffer) |
| Recommended transfer buffer | NuPAGE Transfer Buffer | Bolt Transfer Buffer |
| Gel chemistry | Bis-Tris | Bis-Tris Plus |
| Available polyacrylamide concentrations | 8%, 10%, 12%, 4–12% | |
| Separation range | 15 kDA to 260 kDa (MOPS buffer) 3.5 kDA to 160 kDa (MES buffer) | |
| For use with (equipment) mini gels | Mini Gel Tank or XCell SureLock Mini-Cell | |
| For use with (equipment) midi gels | SureLock Tandem Midi Gel Tank, Invitrogen XCell4 SureLock Midi-Cell or Bio-Rad Criterion (with adapters only) | Not available in midi size |
| Mode of separation | Molecular weight | |
| Application | SDS-PAGE | |
| Available Wells configurations* | Mini: 1, 9, 10, 12, 15, 17, 2D-well, IPG well Midi: 12+2, 20, 26 wells | WedgeWell format: 10, 12, 15 17 well (load up to 2X sample volume per well) |
*Not all percentages are available in every well type
Invitrogen Bis-Tris Gel Highlights
Sharp, straight bands
Neutral-pH buffers in NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels deliver sharp straight bands. During separation, the gels operate close to pH 7. In the Laemmli system (Tris-glycine), the gel is run at basic pH (pH ~9.5). At high pH the residual unpolymerized acrylamide can react with cysteine and lysine residues of the proteins being separated. This may affect downstream analysis such as western blot transfer. At the neutral pH of NuPAGE Bis-Tris and Bolt Bis-Tris Gels, these reactions take place several orders of magnitude more slowly than at the basic pH of Tris-glycine gels, resulting in sharper bands and better resolution.
Protein separation comparing NuPAGE Bis-Tris Gel and traditional tris-glycine gel. The samples listed below were run on (A) a NuPAGE 4–12% Bis-Tris Gel in Invitrogen NuPAGE MES SDS Running Buffer or (B) another manufacturer’s 4–20% tris-glycine gel. Note the high resolution of the sample bands in the NuPAGE protein gel. The poorly resolved “fuzzy” bands seen in the other manufacturer’s gel are a result of reoxidation of some disulfide bonds within the sample, leading to slight changes in migration rates. Lanes 1, 6, 7, 10: Invitrogen Mark12 Unstained Standard; lane 2: high protein load (4 proteins, 6 µg/protein); lane 3: Broad Range Standard; lanes 4, 5: E. coli extract; lane 8: E-PAGE SeeBlue Pre-stained Standard; lane 9: Invitrogen MultiMark Multi-Colored Protein Standard.
Higher-throughput electrophoresis using NuPAGE Bis-Tris Midi Gels
Publication-quality protein electrophoresis data is easily generated using the higher-throughput NuPAGE Bis-Tris mide gels and the SureLock Tandem Midi Gel Tank. The gel produces crisp bands and straight protein lanes.
NuPAGE Bis-Tris 4–12% gradient midi gel result
Publication-quality protein electrophoresis gel results using NuPAGE Bis-Tris 4-12% gradient midi gels and SureLock Tandem Midi Gel Tank. Samples were loaded as follows: Lanes 1, 20: 5 µL PageRuler Broad Range Unstained Protein Ladder; lanes 2–7: 10 µg, 8 µg, 6 µg, 4 µg, 2 µg, 1 µg HeLa lysate, respectively; lanes 8, 13: 5 µL Novex Mark12 Unstained; lanes 9–12: 240 ng, 180 ng, 120 ng, 60 ng of protein mix containing β-galactosidase, lactate dehydrogenase, and lysozyme; lanes 14–19: 10 µg, 8 µg, 6 µg, 4 µg, 2 µg, 1 µg E. coli lysate, respectively. Electrophoresis was conducted with NuPAGE MOPS running buffer and the gel was stained with SimplyBlue Safe Stain.
Better sample integrity
Sample preparation is a crucial step in successful protein analysis. The pH of the traditional Laemmli-style sample buffer changes from 6.8 to 5.2 when heated at 100°C. This lower pH is known to induce aspartyl-prolyl (Asp-Pro) peptide bond cleavage, which leads to protein degradation. By maintaining a >7.0 pH environment, the Invitrogen NuPAGE LDS Sample Buffer and Bolt LDS Sample Buffer preserve protein integrity by minimizing this Asp-Pro cleavage. As seen below, the sample integrity is maintained throughout electrophoresis with the Invitrogen NuPAGE system; protein degradation is seen in samples prepared with Laemmli (Tris-glycine) sample buffer. The Invitrogen NuPAGE Antioxidant running buffer additive greatly minimizes protein oxidation during electrophoresis and keeps reduced protein bands sharp and clear.
High sample integrity. Integrity of samples is maintained throughout electrophoresis with the NuPAGE Bis-Tris Gel System (left), compared to samples prepared with Laemmli (tris-glycine) sample buffer (right).
Bolt Bis-Tris Plus Gel high capacity WedgeWell sample wells
The unique wedge-shaped well in every Bolt gel provides higher loading capacity, so you can load up to twice as much protein sample in every well.
| Well size | Recommended loading volume | Maximum loading volume | Maximum protein load |
|---|---|---|---|
| 10-well | 40 µL | 60 µL | 0.5 µg/band |
| 12-well | 30 µL | 45 µL | 0.4 µg/band |
| 15-well | 20 µL | 35 µL | 0.25 µg/band |
| 17-well | 15 µL | 30 µL | 0.2 µg/band |
Detect proteins even in dilute samples or measure expression of low-abundance proteins. With more room to load your sample, you no longer have to worry about sample spillover and cross-well contamination on your western blots. Gel loading is also easier with no need to use special gel-loading pipette tips.
High sample volume capacity of Bolt Bis-Tris Plus gels. (A) Increasing volumes (40–70μL) of a fluorescent protein standard were loaded in every other lane of an Invitrogen Bolt 4–12% Bis-Tris Plus 10-well Gel. (B) Increasing volumes (10–40 μL) of the same protein standard were loaded in every other lane of Bio-Rad's 4–20% 10-well gel. Sample spillover and cross-well contamination is observed as signals from the sample loaded in the adjacent wells.
Comparison of electrophoresis and western blotting of cell lysates using NuPAGE midi gels versus Bio-Rad
The amount of protein you can load into a protein gel well affects the ability to detect the protein following protein gel electrophoresis or western blotting; the more you can load, the easier it is to detect. The protein load capacities of Invitrogen NuPAGE Bis-Tris precast midi gels run in the SureLock Tandem Mid Gel Tank were compared against Bio-Rad Criterion midi gels run in a Bio-Rad Criterion Cell Midi Cell Tank using manufacturer instructions. Decreasing amounts of HEK 293 cell lysate prepared in RIPA lysis buffer (48–0.5 µg total protein) were denatured in the respective manufacturer’s sample buffer and subjected to electrophoresis using manufacturer instructions. The table below lists the samples, protein mass, and %RIPA buffer loaded in each lane.
| Lane | +1 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | +1 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Sample | Sample buffer | iBright Protein Ladder | HEK293 lysate | iBright Protein Ladder | Sample buffer | |||||||||
| Load mass (µg) | - | - | 48 | 40 | 32 | 24 | 16 | 8 | 4 | 2 | 1 | 0.5 | - | - |
| Load vol. (µL) | 3 | 3 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 3 | 3 |
| % RIPA buffer | - | - | 40 | 33.3 | 26.7 | 20 | 13.3 | 6.7 | 3.3 | 1.7 | 0.8 | 0.4 | - | - |
Invitrogen NuPAGE Bis-Tris gels outperformed Bio-Rad gels at higher lysate loads with blots from Bio-Rad gels showing band loss and smearing at higher loads for all targets investigated.
Comparison of protein blotting and western detection of cell lysates
Western blots using NuPAGE 4-12% Bis-Tris midi gels generate sharper bands at greater protein and RIPA lysis buffer loads than Bio-Rad 4–20% TGX midi gels.A NuPAGE 4–12% Bis-Tris midi gel, 12+2 well, was loaded with decreasing total protein amount of HEK293 lysate, subjected to electrophoresis in a SureLock Tandem Midi Gel Tank and transferred onto a 0.45 µm PVDF membrane using the SureLock Tandem Blot Module and the Bio-Rad 4–20% TGX midi gel, 12+2 well, was subjected to electrophoresis in a Criterion Midi Cell Tank and transferred onto a 0.45 µm PVDF membrane using the Criterion Blotter. Both membranes were probed for three targets (vinculin, α-tubulin, and p23) using fluorescent immunodetection. Band loss and smearing can be seen at the higher loads for all targets on the Bio-Rad blot, while the blot for the NuPAGE Bis-Tris gel offers superior protein loading capacity above 24 µg and crisp, bright bands.
For immunodetection: membranes were blocked for 1 hour in 1X Blocker FL Fluorescent Blocking Buffer. For fluorescence: multiplex detection, the membranes were probed overnight with a mixture of primary antibodies diluted in blocking solution: Rabbit-anti Vinculin (1:15,000), Rat anti-α Tubulin (1:5,000)), and Mouse-anti p23 (1:5,000) followed by an incubation with secondary antibodies diluted at 1:5,000 in 1X Blocker FL: Donkey anti-Rabbit Alexa Fluor Plus 680, Donkey anti-Rat Alexa Fluor 488, and Donkey anti-Mouse Alexa Fluor Plus 800 for 1 hour. Membranes were imaged on an iBright Imaging System, with the same time and image adjustment settings for each blot.
NuPAGE Bis-Tris Gels and Bolt Bis-Tris Plus Gels
NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels are precast polyacrylamide gels designed to give optimal separation for a wide range of molecular weight proteins during gel electrophoresis under denaturing conditions. The Bis-Tris gel chemistry provides a neutral-pH environment that minimizes protein modifications, resulting in less protein degradation and sharper bands without typical gel "smiling". preserving protein integrity becomes particularly important when separating low-abundance proteins.
NuPAGE Bis-Tris gels are offered as mini and midi gels, and Bolt Bis-Tris Plus gels are offered as mini gels. Both NuPAGE Bis-Tris gels and Bolt Bis-Tris Plus gels are ideal for western blot analysis and applications such as sequencing and mass spectrometry where protein integrity is crucial and higher sensitivity is required. The unique WedgeWell design of the Bolt Bis-Tris Plus gel sample well allows loading of up to 2x more sample volume than other precast gels and the Bis-Tris Plus chemistry enables Bolt gels to be run in as little as 20 minutes.
Features of the NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels include:
- Better protein integrity—optimized sample preparation process preserves your proteins
- Wide ranges of molecular weight separation—selecting the right gel and running buffer helps to achieve the optimal separation of your proteins
- Faster run times—separation of your proteins can be attained in 20-35 minutes
- Longer shelf life—NuPAGE Bis-Tris and Bolt Bis-Tris Plus gels can be stored for 16 months at room temperature.
- High capacity wells of Bolt Bis-Tris Plus Gels—wedge-shaped wells makes sample loading easier (no specialized pipette tips required) and allow loads of up to two times higher sample volume
NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels are formulated for denaturing gel electrophoresis applications. For optimal sample preparation, we recommend using buffers containing SDS. The gels can be run using MES SDS running buffer to better resolve smaller proteins (1–200 kDa) and MOPS SDS running buffer to resolve medium- to large-size proteins (14–260 kDa).
Bis-Tris Gels use a Bis-Tris discontinuous buffer system involving three ions:
- Chloride (–) is from the gel buffer and serves as a leading ion due to its high affinity to the anode relative 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 in the running buffer. 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 lower pH gel buffer (pH 6.4) and the running buffer (pH 7.3–7.7) results in a significantly lower operating pH of 7 during electrophoresis.
Get started with Protein gels welcome packs
Protein gels welcome packs are a cost-effective way to start using Invitrogen precast protein gels for the first time. Protein gel welcome packs are available for each of the protein gel chemistries and percentages and come with mini or midi gels, buffers, ladders, and a Mini Gel Tank or SureLock Tandem Midi Gel Tank to provide you with great savings over the cost of the individual components.
Optimized performance of NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels
Unlike traditional Tris-glycine gels, NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels are Bis-Tris HCl buffered (pH 6.4) and have an operating pH of about 7.0. This neutral pH paired with a unique, gentle sample preparation protocol means your protein samples always reside in mild, nonacidic conditions, preserving the integrity of the proteins and minimizing protein modifications. This helps to ensure highly sensitive, accurate western blot results every time.
Greater sensitivity with Bolt Bis-Tris Plus gels. Total cell extracts from A431 cells were transferred to NC and PVDF membranes from a 4–12% Bolt Bis-Tris Plus gel, and 4–20% Tris-glycine precast gel using the iBlot 2 Gel Transfer Device. The cells were treated with 100 ng/mL of human epidermal growth factor (hEGF) to up-regulate expression of the phospho-EGF receptor. The protein loads of the cell extracts ranged from 20 μg to 1.2 μg of extract. The blots were processed on the iBind Western System using a 1:200 dilution of Phospho-EGF Receptor (Tyr1068) (1H12) Mouse mAb (Cell Signaling Technology) and a 1:2,000 dilution of anti-mouse HRP secondary antibody.
Band integrity
NuPAGE and Bolt gels preserve protein integrity with neutral-pH gel chemistry. This formulation minimizes protein modifications during electrophoresis.
A western blot of a Bolt Bis-Tris Plus gel shows clean, sharp protein signals corresponding to only full-length proteins, whereas a western blot of a Bio-Rad TGX gel shows multiple low molecular weight degradation products. Protein kinases implicated in cancer (IKKϐ, EPHB3, HCK, MAPK14, FLT1 and DDR2) were analyzed on a Bolt Bis-Tris Plus Gel and a Bio-Rad TGX tris-glycine gel. The purified kinases (50 ng each), along with MagicMark protein standard and purified recombinant GST protein, were loaded on a 10-well, 4–12% Bolt gel and a 10-well, 4–20% Bio-Rad TGX gel. The samples were separated and transferred to 0.45-μm PVDF membranes using the respective manufacturers’ protocols. Immunodetection was performed using an anti-GST antibody and WesternBreeze chemiluminescence detection.
Band resolution
NuPAGE and Bolt Bis-Tris Plus Gels have better resolving power than Bio-Rad’s gels. With 10% greater resolving distance, optimized gel chemistry, and gradient format, you can see more protein bands resolved on a Bolt gel. In experimental analysis, you can see two separate bands for the insulin B chain and insulin A chain, which have similar molecular weights. The protein bands on the Bolt gel appear straight and the signal intensities are even across the entire bands, whereas the protein bands on Bio-Rad’s gel are uneven and of poor quality.
Bolt Bis-Tris Gel band resolution—comparison of Bio-Rad's stained protein gels.(A) Bolt Bis-Tris gradient gel stained with Invitrogen SimplyBlue SafeStain. (B) Bio-Rad's tris-glycine gradient gel stained with competitor’s stain. (C) Enlargement of a section of lane 5 from the two gels shows the insulin B chain (3.5 kDa) and insulin A chain (2.5 kDa).
Use the protein gel selection guide to find the best mini or midi gel for your protein electrophoresis experiment.
Find the best Invitrogen mini or midi gel to replace your current precast gel from another supplier using the protein gel conversion tool.
Currently using Tris-glycine or Tricine gels? Use the NuPAGE Gel conversion guide below to find the appropriate NuPAGE gel and corresponding optimized buffer.
NuPAGE Gel conversion guide
| If you are using: | Then we recommend NuPAGE gel: | Buffer system |
|---|---|---|
| Tris-Glycine gel | ||
| 4% | NuPAGE 3-8% Tris-Acetate | Tris-Acetate |
| 6% | NuPAGE 3-8% Tris-Acetate | Tris-Acetate |
| 10% | NuPAGE 8% Bis-Tris | MOPS |
| NuPAGE 10% Bis-Tris | MOPS | |
| 12% | NuPAGE 10% Bis-Tris | MOPS |
| 14% | NuPAGE 12% Bis-Tris | MOPS |
| 16% | NuPAGE 12% Bis-Tris | MES |
| 18% | NuPAGE 12% Bis-Tris | MES |
| 4-12% | NuPAGE 3-8% Tris-Acetate | Tris-Acetate |
| NuPAGE 4-12% Bis-Tris | MOPS | |
| 4-20% | NuPAGE 4-12% Bis-Tris | MES |
| 8-16% | NuPAGE 4-12% Bis-Tris | MOPS |
| 10-20% | NuPAGE 12% Bis-Tris | MOPS |
| Tricine gel | ||
| 10% | NuPAGE 8% Bis-Tris | MES |
| NuPAGE 10% Bis-Tris | MES | |
| 16% | NuPAGE 4-12% Bis-Tris | MES |
| 10-20% | NuPAGE 4-12% Bis-Tris | MES |
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