NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels

NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels offer:

• Excellent protein integrity—neutral pH helps reduce protein degradation
• Wide range of molecular weight separation—select the right gel and running buffer to achieve the optimal separation of your proteins
• Fast run times—separate your proteins in 20–35 minutes
• Long shelf life—up to 16 months at room temperature
• High-capacity wells—WedgeWell format wellsarewedge-shaped wells that double the well sample loading capacity and make it easy to load your sample (no specialized pipette tips required)

*Not all percentages are available in every well type
**Shelf life varies depending on gel format

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

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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.

Figure 1. 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.

Excellent 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.

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.

Figure 3. 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.

NuPAGE and Bolt gels preserve protein integrity with neutral-pH gel chemistry. This formulation minimizes protein modifications during electrophoresis.

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.

Publication-quality protein electrophoresis data is easily generated using the higher-throughput NuPAGE WedgeWell format Bis-Tris midi gels and the SureLock Tandem Midi Gel Tank. The gel produces crisp bands and straight protein lanes.
 

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WedgeWell format Midi protein gels allow the separation of up to 26 protein samples in the same run, without compromising on sample loading volume. Here are the key benefits of WedgeWell Midi gels:

• Results you can trust—remove run-to-run variability and enhance the reproducibility of your experiments
• More data in less time—obtain the information you need, faster
• Reduced cost—lower cost per sample and reduced running buffer required per sample
• Load more, see more—easier detection of dilute samples and low abundance proteins
• Easy sample loading—reduced sample spill-over and cross-well contamination

NuPAGE Bis-Tris Midi WedgeWellformat gels meet the performance, reliability, and consistency standards Invitrogen precast gels are known for.

See our Protein Gel Performance Guarantee

NuPAGE Bis-Tris Midi WedgeWell format gels offer the same exceptional protein separation of conventional NuPAGE Bis-Tris Midi gels.

NuPAGE Bis-Tris Midi WedgeWell format gels offer increase sample loading capacity without compromising on the performance of the separation.

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.

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.

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