Figure 2. The protein and RIPA buffer load capacity of Novex 4–20% Tris-Glycine Plus, Midi, 12+2 well WedgeWell format gel exceeds that of Bio-Rad gels. After loading and electrophoresis, a Novex 4–20% Tris-Glycine Plus midi, 12+2 well, WedgeWell format gel, and a Bio-Rad 4–20% Criterion TGX , 12+2 well, midi gel were stained with SimplyBlue SafeStain. The Bio-Rad gel succumbs to protein and lysis buffer overload above a load of 20 µg protein, resulting in streaking and smearing. Staining near the wells indicates some protein has had difficulty entering the gel. The Novex 4–20% Tris-Glycine Plus midi gel provides better protein band sharpness and resolution versus the Bio-Rad 4–20% TGX gel under these loading conditions.
Invitrogen Novex Tris-Glycine Gels are polyacrylamide gels based on traditional Laemmli protein electrophoresis. Novex Tris-Glycine Gels offer reproducible separation of a wide range of proteins into well-resolved bands.
Novex Tris-Glycine Gel specifications
Key features of the Novex Tris-Glycine Gels include:
- Excellent separation—well resolved, straight bands
- Flexibility—use for native and denaturing protein gel electrophoresis
- High-capacity wells—WedgeWellformat wellsarewedge-shaped wells that double the well sample loading capacity and make it easier to load your sample (no specialized pipette tips required)
- Exceptional separation of cell lysates—Novex Tris-Glycine gels resolve lysate samples while other suppliers' gels smear
| Available gel sizes | Mini: 8 cm x 8 cm (1.0 mm thick) Midi: 8 cm x 13 cm (1.0 mm thick) |
| Available Well configurations* | WedgeWell format, Midi (load up to 100 µL per well): 12+2, 20, 26 wells WedgeWell format, Mini (load up to 60 µL per well): 10, 12, 15 wells Midi: 12+2, 20, 26 wells |
| Storage conditions | 2–8°C |
| Shelf life | Up to 12 months |
| Recommended sample buffer | SDS-PAGE: Novex Tris-Glycine SDS Sample Buffer Native-PAGE: Novex Tris-Glycine Native Sample Buffer |
| Recommended running buffers | SDS-PAGE: Novex Tris-Glycine SDS Running Buffer Native-PAGE: Novex Tris-Glycine Native Running Buffer |
| Recommended transfer buffers | Novex Tris-Glycine Transfer Buffer |
| Gel chemistry | Tris-glycine |
| Available polyacrylamide concentrations | 6%, 8%, 10%, 12%, 14%, 16%, 4–12%, 4–20%, 8–16%, 10–20% |
| Separation range (denaturing) | 8–250 kDa |
| 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) |
| Mode of separation | SDS-PAGE: Molecular weight Native-PAGE: Intrinsic charge, molecular size |
| Applications | SDS-PAGE, Native-PAGE |
*Not all percentages are available in every well type
Novex Tris-Glycine Gels do not contain SDS and can be used to run your proteins in native or in denatured form. For denatured proteins, we recommend using tris-glycine SDS sample buffer and a tris-glycine SDS running buffer. For native proteins, we recommend using a tris-glycine native sample buffer and a tris-glycine native running buffer.
Novex Tris-Glycine Gels use a tris-glycine discontinuous buffer system with three ions primarily involved:
- Chloride (–), supplied by the gel buffer, serves as the leading ion because it has the highest attraction to the anode relative to other anions in the system
- Glycine (–), the primary anion provided by the running buffer, serves as the trailing ion, because it is only partially negatively charged and remains behind the more highly charged chloride ions in a charged environment
- Tris base (+) is a common ion present in both the gel and the running buffers. During electrophoresis, the gel and buffer ions in the tris-glycine system form an operating pH of 9.5 in the separating region of the gel.
High-capacity wells now also available in the midi gel format
WedgeWell format wells are wedge-shaped wells that increase the sample loading capacity, allowing up to 60 µL and 100 µL of samples to be loaded in the mini and midi gel wells, respectively. WedgeWell Midi format gels that combine the higher sample throughput of wide format gels with the higher sample loading capacity of WedgeWells are now available. Key benefits include:
- Results you can trust—rigorously tested to help ensure reliable, reproducible performance, lot after lot
- Easy sample loading—larger well openings make loading a breeze even with standard pipette tips, and reduce sample spill-over and cross well contamination
- Increased sample loading capacity—easy detection of dilute samples and low abundance proteins
- Reduced cost—lower cost per sample and reduced running buffer required per sample (7.3 mL of running buffer saved per sample, 140 mL saved for 20 samples)*
- Faster run times—up to 10 min faster than traditional gel
Recommended sample loading volumes for Novex Tris-Glycine Gels
| Well size | Maximum loading volume (µL) | |
|---|---|---|
| WedgeWell format midi gels | Standard midi gels | |
| 12 + 2 (small wells) | 100 + 35 | 50 + 15 |
| 20 | 60 | 30 |
| 26 | 40 | 20 |
| WedgeWell format mini gels | ||
| 10 | 60 | |
| 12 | 45 | |
| 15 | 35 | |
| 17 | 30 | |
*1 mm thick gel
Increased sample loading capacity
WedgeWell format well increases the well sample loading capacity, helping prevent sample spillover and cross-contamination.
Figure 1. Increased sample volume capacity of Novex Tris-glycine midi WedgeWell format gels. (A) To compare gel spillover, increasing volumes (20–60 μL) of a fluorescent protein ladder were loaded in every other lane of a Novex WedgeWell Tris-glycine midi 20-well gel (left) or Bio-Rad 4–20% Criterion TGX Protein Gel (right). Sample spillover in Bio-Rad’s gel is seen in lanes adjacent to the 40–60 μL loading lanes.
Comparison of electrophoresis and western blotting of cell lysates using Novex 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 Novex Tris-Glycine Bis-Tris precast midi gels run in the SureLock Tandem Midi 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 Novex Tris-Glycine 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.
| 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) | NA | NA | 60 | 50 | 40 | 30 | 20 | 10 | 5 | 2 | 1 | 0.5 | NA | NA |
| Load vol. (µL) | 5 | 3+25 S.B. | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 3+25 S.B. | 5 |
| Conc. (µg/µL) | NA | NA | 2 | 1.67 | 1.33 | 1 | 0.67 | 0.33 | 0.17 | 0.07 | 0.03 | 0.02 | NA | NA |
Protein gel electrophoresis of cell lysates
Novex Tris-Glycine Plus, Midi, WedgeWell format gels offer increased loading capacity and excellent protein separation.
Comparison of protein blotting and western detection of cell lysates
Figure 3. Western blots using Novex 4–20% Tris-Glycine Plus midi gels display sharper bands at greater protein and RIPA lysis buffer loads than Bio-Rad 4–20% TGX midi gels. A Novex 4–20% Tris-Glycine Plus 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. In parallel, a 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 analyzed for total protein using the No-Stain Protein Labeling Reagent, followed by chemiluminescent immunodetection of three targets: Vinculin, α-Tubulin, and p23. The Bio-Rad blot shows streaking, bowing of bands above 24 µg protein, and bleed-over into the ladder lane for total protein analysis. With immunoblotting, the Bio-Rad blot shows band loss and smearing at higher loads for all targets. For immunodetection: membranes were blocked for 1 hour in 1X Blocker FL Fluorescent Blocking Buffer. For chemiluminescent detection: the membranes were probed overnight with a mixture of primary antibodies diluted in blocking solution: Rabbit-anti Vinculin (1:30,000), Rat anti-α Tubulin (1:15,000), and Mouse-anti p23 (1:60,000) followed by an incubation with secondary antibodies in 1X Blocker FL: Donkey anti-Rabbit HRP (1:5,000), Donkey anti-Rat HRP (1:30,000), and Donkey anti-Mouse HRP (1:240,000) for 1 hour. Membranes were incubated for 5 minutes with SuperSignal West Dura Extended Duration Substrate and imaged for the same amount of time on an iBright Imaging System.
Novex Tris-Glycine Gels—excellent protein band resolution
Novex Tris-Glycine Gels are designed to deliver well-resolved, straight bands with optimal band quality as compared to other commercially available precast gels.
Novex Tris-Glycine Gel band quality
Novex Tris-Glycine Gels are designed to deliver well-resolved, straight bands with optimal band quality as compared to other commercially available precast gels.
Figure 4. Band quality with Novex Tris-Glycine gels. Protein ladders, purified proteins, and E. coli lysate were loaded on an Invitrogen Novex 4–20% Tris-Glycine Mini Gel, (A) and a 4–20% gradient gel (B). Straighter lanes with better lysate protein band sharpness and resolution are observed on gel (A). Lanes 1, 5, 10: 5 µL Thermo Scientific PageRuler Unstained Protein Ladder; lanes 2, 6, 9: 5 µL Invitrogen Mark12 Unstained Standard; lane 3: 10 µg E. coli lysate; lane 4: 6 µg BSA; lane 7: 6 µg hIgG; lane 8: 20 µg E. coli lysate.
Figure 5. Novex Tris-Glycine Gels deliver sharp straight bands. Protein ladders and A431 cell lysate were loaded on a Novex Tris-Glycine Gel, 4–20% gradient and transferred to nitrocellulose using the Invitrogen iBlot 2 Gel Transfer Device. Lane 1: Invitrogen iBright Prestained Protein Ladder; Lane 2: Invitrogen MagicMark XP Western Protein Standard; Lanes 3–7: A431 cell lysate, 15 µg, 5 µg, 1.67 µg, 0.55 µg, 0.19 µg.
Protein integrity
With Novex Tris-Glycine Gels, you can achieve greater protein integrity as compared to other commercially available precast gels.
Figure 6. Novex Tris-Glycine Gels offer increased protein integrity. Protein ladder, purified proteins, and E. coli lysate were loaded on a Novex 4–20% Tris-Glycine Mini Gel,(A) and a Bio-Rad TGX 4–20% gradient gel (B). The Bio-Rad TGX gel (B) displays numerous low molecular weight protein degradation products below major bands in lanes 3, 4, 7, 8. These are not seen in the Novex Tris-Glycine gel (A). Gel (A) also displays better lysate protein band sharpness and resolution than gel (B). Lanes 1, 10: 5 µL Mark12 Unstained Standard; lane 2: 10 µg E. coli lysate; lane 3: 6 µg catalase; lane 4: 6 µg carbonic anhydrase; lane 5: 6 µg lysozyme; lane 6: 6 µg hIgM ; lane 7: 6 µg BSA; lane 8: 6 µg beta-galactosidase; lane 9: 20 µg E. coli lysate.
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