Specialized Protein Gels Support — Getting Started
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The recommended storage temperature for Novex™ Tricine gels is 4 degrees C where the shelf life varies from 4–8 weeks depending upon the gel percentage. The higher the percentage, the shorter is the shelf life.
Novex™ Tricine Gels are ideal for peptides and low molecular weight proteins (less than 10 kDa). Unlike Tris-Glycine gels, Tricine gels allow resolution of proteins with molecular weights as low as 2 kDa. Tricine, unlike glycine, will not interfere with sequencing, so Tricine gels are an excellent choice for direct sequencing after transferring to PVDF. In addition to good transfer efficiency, the Tricine system has a lower pH which minimizes unwanted protein modification. Tricine gels can only be run under denaturing conditions.
Tricine gels do not contain SDS. The Tricine system requires SDS in the sample and running buffers for best results. They are run using the Tricine SDS Sample buffer and Tricine SDS Running buffer.
Tricine gels contain a 4% stacking gel that is ~8 to 9 mm long.
The ratio of acrylamide:bisacrylamide in our Tricine gels is 37.5:1 and percentage of crosslinker is 2.6%.
The Tricine gel system, first described by Schagger and von Jagow in 1987, is a modification of the Laemmli Tris-Glycine system to allow for better resolution of smaller proteins and peptides. In the Laemmli system, the proteins are "stacked" in the porous, top portion of the gel (stacking gel) between the highly mobile "leading" chloride ions, present in the gel buffer and the slower "trailing" glycine ions, supplied by the running buffer. These stacked protein bands undergo sieving once they reach the separating gel, thus resolving by size. However, the resolution of smaller proteins (under 10 kDa) is hindered by the continuous accumulation of free dodecyl-sulfate (DS) ions (from the SDS sample and running buffers) in the stacking gel. This build-up of DS leads to convective mixing of the DS ions with the smaller proteins, causing fuzzy bands and decreased resolution. The mixing of the DS ions with the small proteins also interferes with the fixing and staining process later.
To solve this problem, we offer the Novex™ Tricine gel system that is based on the Tris-Glycine system developed by Schagger and von Jagow. This modified system uses a low pH in the gel buffer and substitutes the trailing glycine ions with faster moving tricine trailing ions. Many small proteins and peptides that migrate with the stacked DS micelles in the Tris-Glycine system are now well separated from DS ions in the Tricine gel system, resulting in sharper, cleaner bands and higher resolution.
No, the Tricine is actually supplied by the running buffer.
Yes. Tricine, unlike glycine, does not interfere with sequencing reagents.
Adding urea to the sample and running buffers, in conjunction with SDS, may provide improved solubilization of the sample if denaturation by SDS does not prove to be sufficient. This must be tested empirically for the protein of interest.
For blotting Tricine gels, we recommend using 1X Tris-Glycine Transfer Buffer with 20% methanol. The Tris-Glycine Transfer Buffer interferes with protein sequencing. Hence, if you are performing protein sequencing, we recommend using a non-glycine based transfer buffer such as 1X NuPAGE™ Transfer Buffer, 0.5X TBE Transfer Buffer or CAPS buffer (10 mM CAPS (3 cyclohexylamino, 1-propanesulfonic acid), 10% methanol, pH 11.0).
The Novex 10% Zymogram Gelatin gels are 10% Tris-Glycine gels with 0.1% Type A gelatin from porcine skin (Sigma Cat. No. G2500), as the substrate.
The Novex™ 12% Zymogram Casein gels are 12% Tris-Glycine gels with 0.05% casein as the substrate. The casein is ~26 kDa in size. The type of casein used is proprietary.
Novex™ 4–16% Zymogram Blue-Casein gels are 4–16% Tris-Glycine gels with 0.1% casein that is covalently linked to a proprietary dye, as the substrate. The casein is ~26 kDa in size. The type of casein used is proprietary. The presence of the dye makes visualization easy without having to stain the gel.
Our Zymogram gels do not contain SDS. We recommend using the Novex™ Tris-Glycine SDS Sample buffer and the Novex™ Tris-Glycine SDS Running buffer with these gels. Novex™ Zymogram Renaturing buffer and Novex™ Zymogram Developing buffer are recommended for enzyme assays.
The ratio of acrylamide:bisacrylamide in our Zymogram gels is 37.5:1 and percentage of cross-linker is 2.6%.
Yes, our Zymogram gels do contain a 4% stacking gel that is ~8 to 9mm long.
Our Zymogram gels do not contain the substrate (gelatin or casein) in the stacking gel although a small amount might mix in during casting of the gel.
We recommend storing the Zymogram gels at 4 degrees C.
The shelf life of Zymogram gels is 8 weeks when stored at 4 degrees C.
The Zymogram buffers should be stored at 4 degrees C.
Novex™ Zymogram gels are an excellent tool for detecting and characterizing proteases that can utilize casein or gelatin as a substrate. Proteases are run on a gel containing one of these substrates under SDS conditions. They can be visualized as clear bands against a dark background following a simple renaturing and staining protocol. These gels are commonly used to detect matrix metalloproteases, a family of enzymes linked to the pathogenesis of cancer and some inflammatory diseases.
The substrates for the matrix metalloproteases (MMPs) are:
MMP-1: tissue collagenase: collagen 1, 2, 3, 4, 6, & 10
MMP-2: gelatinase: gelatins, collagens 4, 5, & 7
MMP-3: stromleysin-2: casein, fibronectin, laminin, elastin
MMP-7: matrilysin: casein
MMP-8: neutrophil collagenase: collagen
MMP-9: type 4 collagenase: gelatin
MMP-10: stromelysin-2: casein
MMP-12: metalloelastase: elastin
No, samples should not be heated or reduced so that multiunit proteases migrate as a single unit that can be renatured after electrophoresis. The sample should be one part sample and one part 2X Tris-Glycine SDS Sample buffer. Let the sample stand at room temperature for 10 minutes and then load the sample onto the gel.
Zymogram gels are essentially Tris-Glycine gels containing the substrate. Protein standards run based solely on the percentage of acrylamide and hence should run the same in both kinds of gels. It is quite possible though that if the standard is pre-stained, the proteins will appear a different color because of the staining (or pre-staining) of the Zymogram gels.
We decided to go with 0.05% casein because it gives higher sensitivity. 0.1% casein and 0.2% casein provide higher contrast of the bands but lower sensitivity because the enzyme has to digest more substrate in order to form the clear band.
The casein in the 4–16% Zymogram Blue-Casein gels is covalently bound to a dye. Some enzymes have a harder time digesting the dye-bound casein compared to the unmodified casein and also it takes more activity to clear the higher quantity of casein.
Zymogram gels can be stained using Colloidal Blue stain or SimplyBlue™ SafeStain. The fixing step is not required. We recommend staining with SimplyBlue™ SafeStain after renaturing and developing the gel for enzyme activity. If greater contrast is needed for detecting weakly active enzymes, a 0.5% Coomassie™ R250 stain may be used.
The NativePAGE™ 5% G-250 Sample Additive (Cat. No. BN2004) is a concentrated stock solution of Coomassie™ G-250 designed for use with detergent (non-ionic) containing samples prepared for NativePAGE™ gel electrophoresis. The exact composition of the NativePAGE™ 5% G-250 Sample Additive is proprietary. It does not contain a detergent.
The NativePAGE™ 5% G-250 Sample Additive is a concentrated stock solution of Coomassie™ G-250 designed for use with detergent (non-ionic) containing samples prepared for NativePAGE™ gel electrophoresis. Native proteins normally migrate on the gel according to natural charge/pI in the pH of gel buffer, but adding the Coomassie™ G-250 renders an associated negative charge even to high pI proteins that would normally have a positive charge. It binds to proteins non-specifically without denaturing them. The NativePAGE™ 5% G-250 Sample Additive is added to the cathode buffer and, optionally, to samples as well.
Our NativePAGE™ gels do have a stacking gel. The stacking gel for these gels is a ~1 cm region at the top of the gel where the acrylamide percentage is low (4%) and constant. Below the stacking gel, the acrylamide percentage begins to increase in the gradient portion of the gel. However, the gel buffer is the same throughout the gel. So the stacking gel in NativePAGE gels is not the same as in the Laemmli system where the stacking gel has a different pH causing a decreased ion mobility for the trailing ions. Also the entire NativePAGE gel is cast in one continuous delivery due to which no demarcation line is seen between the resolving (or gradient) portion of the gel and the stacking gel.
We recommend storing them at 4–25 degrees C. They should not be frozen.
The NativePAGE™ Novex™ Bis-Tris Gels have a wide range of separation throughout the low- and high–molecular weight ranges:
- The NativePAGE™ Novex™ 3–12% Bis-Tris Gels resolve proteins in the molecular weight range of 30–10,000 kDa.
- The NativePAGE™ Novex™ 4–16% Bis-Tris Gels resolve proteins in the molecular weight range of 15–10,000 kDa.
NativePAGE™ Sample Buffers and Running buffers were developed specifically for use with the NativePAGE™ Bis-Tris gels. We do not recommend using these buffers for native applications with any other gels, including NuPAGE™ Tris-Acetate or Novex™ Tris-Glycine gels. For those gels, we recommend using the Novex™ Tris-Glycine Native Sample and running buffers.
We do not recommend using NativePAGE™ Sample and Running buffers with NuPAGE™ Bis-Tris gels. NuPAGE™ Novex Bis-Tris gels are optimized for denaturing conditions and have an extremely low operating pH (pH 7.0), which makes it difficult for most proteins to migrate through them in a native state.
Samples requiring non-ionic detergents for solubility are not compatible with traditional native Tris-Glycine PAGE because as the proteins migrate into the polyacrylamide gel, they leave behind the non-ionic detergents. In the absence of non-ionic detergent, the proteins aggregate and form vertical streaks at the top of the lane. When using blue native electrophoresis (NativePAGE™ gels), the Coomassie™ G-250 dramatically reduces aggregation to allow the resolution of membrane protein complexes not seen in the Tris-Glycine gel.
In addition, compared to the operative pH of the Tris-glycine system (pH 9.3–9.5), the lower operative pH of the NativePAGE™ gels (pH 7.5–7.7) may help to retain the native structure and/or activity of proteins sensitive to alkaline pH.
NativePAGE™ gels have been successfully used to perform EMSA showing protein-protein interactions between two purified proteins, but we have not tested NativePAGE™ gels for EMSA analysis of interactions between nucleic acids (either DNA or RNA) and a protein or protein complex.
We recommend using the NativeMark™ Unstained protein standard, Cat. No. LC0725.
No, most of our protein standards (including the Novex™ Sharp prestained and unstained standards) are already denatured and reduced, and will not resolve well in a native gel. For NativePAGE™ electrophoresis, we offer the NativeMARK™ Unstained Protein Standard (Cat. No. LC0725). It is a ready-to-use protein marker that allows for molecular weight estimation of proteins using native gel electrophoresis.
Please note that even with a native standard, molecular weight estimation in native electrophoresis is very inaccurate, as gel migration can be significantly affected by differing charge and conformation of individual proteins. For more accurate estimations, use SDS-PAGE separation or mass spectrometry analysis.
NativePAGE™ Novex™ Bis-Tris Gels are compatible with most of the standard Coomassie™ R-250 or G-250 staining formulations. The Novex™ Colloidal Blue Staining Kit (Cat. No. LC6025) is recommended for the most sensitive Coomassie™ staining in NativePAGE™ Gels. SimplyBlue™ Safe Stain (Cat. No. LC6060) is not recommended for use with NativePAGE™ gels, due to special fixing requirements. Both the SilverQuest™ Silver Staining Kit (Cat. No. LC6070) and SilverXpress™ Silver Staining Kit (Cat. No. LC6100) are suitable for staining of NativePAGE™ Gels. However, for best overall results and lower background, we recommend the SilverQuest™ Kit as the best choice.
We recommend using the NuPAGE™ Transfer buffer for blotting of NuPAGE gels. PVDF is the recommended blotting membrane and works well in terms of transfer and detection. Nitrocellulose is not compatible with blotting of NativePAGE™ Gels, as the nitrocellulose membrane binds the Coomassie™ G-250 dye very tightly and is not compatible with alcohol-containing solutions needed to destain the membrane and fix the proteins.
Although the gels share some major chemical components, they are not interchangeable. NuPAGE™ Bis-Tris gels are optimized for denaturing and reducing conditions, and their neutral pH makes it difficult to use them for native applications as most proteins will have no charge or positive charge. Therefore, native applications with these gels are not recommended. NativePAGE™ Bis-Tris gels on the other hand have a different formulation that has been optimized for native electrophoresis with highest resolution. The buffers for the two types of gels should not be interchanged.
E-PAGE™ gels are self-contained, pre-cast gels (neutral pH) that include a buffered gel matrix and electrodes packaged inside a disposable, UV-transparent cassette. They are designed for fast, buffer-less, high-throughput protein electrophoresis in a horizontal format and are available in 96-well, 6% and 48-well, 8% formats. Each E-PAGE™ 96 6% gel contains 96 sample lanes and 8 marker lanes in a patented staggered well–format that is compatible with the standard 96-well plate format for loading with a multichannel pipette or with an automated liquid handling system. Each E-PAGE™ 48 8% gel contains 48 sample wells and 4 marker wells. The wells are compatible for loading with a multichannel pipette in alternating lanes or with an automated liquid handling system.
The gel formulation of E-PAGE™ gels is proprietary.
E-PAGE™ gels do not contain a preservative. They undergo UV sterilization during the production process.
The molecular weight range of proteins that can be separated is 10–220 kDa for E-PAGE™ 96 6% gels and 10–200 kDa for E-PAGE™ 48 8% gels.
The running distance of each lane is 1.6 cm for E-PAGE™ 96 6% and 3.2 cm for E-PAGE™ 48 8% gels.
E-PAGE™ gels contain SDS and are recommended for performing electrophoresis under denaturing conditions.
We recommend loading 1–20 μg protein per lane of an E-PAGE gel. The maximum recommended protein load per lane gel is 20 μg. Excess protein will cause poor resolution and smearing.
The recommended total sample loading volume for E-PAGE™ 48 and E-PAGE™ 96 gels is 15 µL (up to 20 µL). Very low volumes of sample loaded will result in poor resolution and smearing. For proper band separation, we recommend keeping sample volumes uniform and loading deionized water into the empty wells.
We recommend using the 4X E-PAGE™ Loading Buffer 1 (supplied with E-PAGE™ gels) for preparing samples for SDS-PAGE and staining or blotting using E-PAGE™ gels. We do not recommend using any other SDS sample buffer such as the NuPAGE™ LDS sample buffer or the Tris-Glycine SDS sample buffer as there may be a loss of resolution in the bands.
We recommend using the Lumio gel sample buffer that is supplied with the Lumio Green Detection kit, for in-gel detection. We do not recommend using the E-PAGE loading buffer. There is no need to remove the E-PAGE gels from the cassette to visualize Lumio fusion proteins.
The composition of the 4X E-PAGE™ Loading Buffer 1 is proprietary.
We recommend using the Large Gel Drying Kit (Cat. No. NI2207) for drying E-PAGE™ gels. It is also possible to air dry E-PAGE™ gels. The E-PAGE™ 96 gels will need at least 4 days for complete air drying. Vacuum drying is not recommended—the thickness of these gels makes them especially prone to cracking.
The recommended run time for E-PAGE™ 96 6% gels is 14 minutes and for E-PAGE™ 48 8% gels is 25 minutes. No pre-run is necessary. It is possible to run longer than the recommended run time to improve resolution, keeping in mind the risk of running into the next well below. Avoid running E-PAGE™ 96 or 48 gels for more than 25 and 30 minutes respectively. Running power is 9.5 watts.
Please see the table below for the recommended protein standards:
|Application||Recommended protein standard
|E-PAGE™ 48 8% gels
||E-PAGE™ 96 6% gels|
|Electrophoresis||SeeBlue™ Plus 2 Pre-Stained Standard, Cat. No. LC5925
||E-PAGE™ SeeBlue™ Pre-Stained Standard, Cat. No. LC5700|
|Western blotting||MagicMark™ XP Western Protein Standard, Cat. No. LC5602
||E-PAGE™ MagicMark™ Unstained Protein Standard, Cat. No. LC5701|
|Fluorescence detection||BenchMark™ Fluorescent Protein Standard, Cat. No. LC5928||BenchMark™ Fluorescent Protein Standard, Cat. No. LC5928|
The Mother E-Base™ device (Cat. No. EB-M03) comes with a power cord that can be connected directly to an electrical outlet and is used for electrophoresis of one E-PAGE™ gel. The Daughter E-Base™ device (Cat. No. EB-D03) connects to the Mother E-Base™ and to other Daughter E-Base™ devices for the simultaneous electrophoresis of two or more gels. The Daughter E-Base™ device does not connect with a power cord and cannot be used without a Mother E-Base™ device. The Mother E-Base™ has been tested for electrophoresis with up to three Daughter E-Bases™ connected at one time.
After electrophoresis is completed, the E-PAGE™ gel cassette can be opened with the Butterfly Opener (supplied with E-PAGE™ gels) to perform downstream applications such as western blotting or staining.
E-PAGE™ gels are compatible with many standard Coomassie™, silver, or fluorescent staining protocols. E-PAGE™ gels are thicker than most SDS-PAGE mini-gels, so additional time may be required for staining and destaining steps. We recommend the following stains for E-PAGE™ Gels. Detailed staining protocols are described on pages 40–49 of the E-PAGE™ Technical guide.
Total protein stains:
- SYPRO™ Ruby Protein Gel Stain (page 40)
- Coomassie™ Stain (page 42)
- SimplyBlue™ SafeStain (page 44)
- SilverQuest™ Silver Staining Kit (page 47)
- SilverXpress™ Silver Staining Kit (page 48)
Specific protein stains:
- Lumio™ Green Detection Reagent for detecting Lumio fusion proteins (page 40)
- InVision™ His-tag In-Gel Stain for detecting 6X His-tagged proteins (blotting recommended first, page 49)
We recommend using the EP program for running E-PAGE™ gels.
The E-Holder™ Platform is designed to hold E-PAGE™ Gels during loading. You can use the E-Holder™ Platform when you need to load multiple gels while other gels are running on the E-Base™ device.
Note: The E-Holder™ Platform is not a power supply unit, cannot be connected to an electrical outlet, and cannot be used to run E-PAGE™ 48 or 96 Gels. To obtain the best results, run E-PAGE™ Gels on the Mother E-Base™ Device or Daughter E-Base™ Device within 15 minutes after loading on E-Holder™ Platform.
We recommend keeping the surfaces of the Mother E-Base™ Device and Daughter E-Base™ Device free of contaminants. To clean, disconnect bases from power source and wipe with a dry cloth. Do not attempt to open or service the bases.
For Research Use Only. Not for use in diagnostic procedures.