Technology

Our Technology

PAGEgel products represent the first significant technological and convenience advancements in vertical gel electrophoresis in over a decade. Improvements in gel technology, buffer formulations and cassette design produce high-performance separations in an easy-to-use product.

Gel Technology Improvements

PAGEgel has developed a proprietary photo-polymerization process. Using a novel photo-initiator, PAGEgel is able to manufacture composite-polymer gels (primarily cross-linked polyacrylamide with a small amount of agarose) in a process that allows gel solutions to be stored at high temperatures and gels cast without polymerizing the acrylamide.

In the PAGEgel process, the gels are cast, the agarose is allowed to set, and then the acrylamide is photo-polymerized using proprietary methods to generate controlled rates of polymerization throughout the gel. This process produces maximum resolution, smoothes the acrylamide gradient, and eliminates any casting defects. It also allows us to polymerize gel fingers that protrude above the cassette.

Normally, polyacrylamide wants to swell up to 30% after polymerization, which disrupts resolution and gel straightness. The composite-polymer gels confer a number of advantages, such as:

• Incredible gel strength and elasticity to simplify handling and processing gels;
• A 10 to 20-fold reduction in gel swelling, creating superior physical stability, even at high gel percentages;
• Extends the polyacrylamide porosity range at low percentages, which produces more efficient stacking for larger sample volumes, and permits separation of larger proteins (up to 1,000kDa).

Buffer Technology Improvements

PAGEgel's neutral pH gel and running buffer system to add chemical stability to the physical stability produced by the composite polymer. The lower gel buffer pH slows base-catalyzed acrylamide hydrolysis 10-fold over the classic Laemmli system (Tris chloride at pH 8.6 to 8.8).

For the SDS gel line, the gel buffer is triethanolamine chloride at pH 7.6, along with 0.1% SDS. Since SDS is required in composite-polymer gels for SDS PAGE, an alternate gel buffer system was developed for native proteins and DNA. The DNA/Native gels employ Tris acetate (pH 7.6) as the buffer. Acetate is used as the anion to produce sharp DNA bands, and accommodates native protein separations, too. Combined with the composite polymer, PAGEgel gels are stable and have been demonstrated to work successfully for years at 4°C, and for months at room temperature.

Well Technology Improvements

In typical electrophoresis gels, once the wells are loaded with water or running buffer, they virtually disappear. PAGEgel incorporates inert colored plastic beads (patent pending) into the gel loading area. The colored beads highlight the wells and make it easy to see the wells. Different colors are used with different gel buffer systems to make it clear the appropriate running buffer for that particular gel.

Cassette Technology Improvements

PAGEgel has added a convenient feature to the typical plastic cassette. The cassettes are made with an interference fit to make them easy to open. Notches in the shorter plate line up with the gel fingers to lock them in place. Additionally, due to our polymerization technology, no coatings are needed on the cassette interior to get high-resolution separations.

Electrophoresis Background

Gel electrophoresis is a technique used for the separation of DNA, RNA, or protein molecules using an electric current applied to a gel matrix. "Electrophoresis" refers to the electromotive force (EMF) that is used to move the molecules through the gel matrix. By placing the molecules in wells in the gel and applying an electric current, the molecules will move through the matrix at different rates, usually determined by mass, toward the positive anode if negatively charged or toward the negative cathode if positively charged.

In the case of nucleic acids, the direction of migration, from negative to positive electrodes, is due to the naturally-occurring negative charge carried by their sugar-phosphate backbone. Double-stranded DNA fragments behave as rods, so their migration through the gel is relative to their structure, for non-cyclic fragments, size. Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that linearize (single-stranded) or disrupt the hydrogen bonds (double-stranded), such as restriction enzymes, sodium hydroxide or formamide, are used to cut or denature the nucleic acids and cause them to behave as rods.

Proteins, unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate into the gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are usually denatured in the presence of a detergent such as sodium dodecyl sulfate (SDS) that coats the proteins with a negative charge. In general, the amount of SDS bound is relative to the size of the protein, so that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Because the proteins have the same charge-to-mass ratio, and because the gels have sieving properties, mobility becomes a function of molecular weight.

The velocity of a charged particle moving in an electric field is directly proportional to the field strength and the charge on the molecule and is inversely proportional to the size of the molecule and the viscosity of the medium. Adding a gel with sieving properties (that is a gel where the resistance to the motion of a particle increases with particle size) increases the differences in mobility between proteins of different molecular weights. This is the basis of separation.

The original use of gels as separating media involved using a single gel with a uniform pH throughout. Molecules were separated on the basis of their mobility through a single gel matrix. This system has only occasional use in today's laboratory. It has been replaced with discontinuous, 4 multiple gel systems. In multiple gel systems, a separating gel is augmented with a stacking gel and an optional sample gel. These gels can have different concentrations of the same support media, or may be completely different agents. The key difference is how the molecules separate when they enter the separating gel. The proteins in the sample gel will concentrate into a small zone in the stacking gel before entering the separating gel. The zone within the stacking gel can range in thickness from a few microns to a full millimeter. As the proteins are stacked in concentrated bands, they continue to migrate into the separating gel in concentrated narrow bands. The bands then are separated from each other on a discontinuous pH gel.

The separation of molecules within a gel is determined by the relative size of the pores formed within the gel. The pore size of a gel is determined by two factors, the total amount of acrylamide present and the amount of cross-linker. As the total amount of acrylamide increases, the pore size decreases.

In the traditional Laemmli method, when a current is added, Glycine is a weak acid and it can exist in either of two states, an uncharged zwitterion, or a charged glycinate anion (negatively charged). At low pH it is protonated and thus uncharged. At higher pH it is negatively charged. When the power goes on the glycine ions in the running buffer want to move away from the cathode (the negative electrode) so they head toward the sample and the stacking gel. The pH there is low and so they lose a lot of their charge and slow down. Meanwhile, in the stacker and sample the highly mobile chloride ions (which are also negatively charged) move away from the cathode too. This creates a narrow zone of very low conductance in the top of the stacking gel. The very high field strength makes the negatively charged proteins move forward. The caveat is that they can never outrun the chloride ions or they would find themselves in a region of high conductance and very low field strength and would immediately slow down. The result is that all the proteins move through the stacker in a tight band just behind the moving front of chloride ions. Behind them, the glycine ions move along with lower mobility than the chloride ions. The effect of this moving zone of high voltage is that all the proteins reach the running gel at virtually the same time so that migration of the proteins is truly a function of molecular size and not some complicated function of how carefully you loaded the gel and when you started the voltage.

When the ions hit the running gel the pH goes way up and the glycine becomes deprotonated (and thus more negatively charged). The mobility of the glycine goes way up and the mobility of the proteins goes way down (due to the sieving properties of the gel). The result is that the glycine races past the protein and the proteins are no longer in a narrow zone of very high resistance (and very high electric field). The proteins now move along in a uniform electric field and separate based on size.