Size-base separation

Size-based analysis of SDS-protein complexes in polyacrylamide gels (SDS-PAGE) is the most common type of slab gel electrophoresis for the characterization of polypeptides, and SDS-PAGE is one of the most commonly used methods for the determination of protein molecular masses.117 The uses for size-based techniques include purity determination, molecular size estimation, and identification of posttranslational modifications.118119 Some native protein studies also benefit from size-based separation, e.g., detection of physically interacting oligomers. 

The CE-SDS method is a size-based separation technique generally applicable to proteins from 10 to -200—300kDa. The specificity is generally tested against the formulation buffer and any other possible contaminant proteins. There is usually no interference from the formulation buffer with the assay. For samples that contain contaminant proteins with a hydrodynamic size of 10—200kDa, the method is not specific. 

Nowadays, the sieving matrices most employed in CSE are polymer solutions that under suitable conditions may form a transient mesh or sieving matrix that provide the size-based separation of charged biopolymers. The polymer solutions can be formulated with linear acrylamide and N-substituted acrylamide polymers, cellulose derivatives, polyethylene oxide, and its copolymers or with a variety of polymers, such as polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), and hydroxypropyl cellulose(HPC), which do not necessitate the preventive coating of the capillary wall due to their ability to dynamically coat the inner surface of the capillary, resulting in suppressed EOE and sample interactions with the capillary wall. 

In FFF systems, the separation along the flow axis is caused by the perpendicular field, whose crucial role is recognized by the word "field" 1n field-flow fractionation. The applied field Interacts with entrained particles, forcing them to accumulate at one wall (the accumulation wall) of the channel. Since the flow velocity near any wall is reduced by frictional drag, the downstream displacement of the particles 1s retarded. Retardation (or retention) is greatest for those particles forced most closely to the wall. Consequently, particles are separated according to the different forces exerted on them by the applied field. These forces normally depend on particle size, leading to a size-based separation. 

Eq. 8.19, is largely independent of size for most colloidal particles of like composition, making the size-based separation of colloids by electrophoresis difficult. Similarly, the mobility of uniformly charged polymer chains (such as DNA and SDS denatured proteins) is nearly independent of chain length in free solution. 

Zhu, M., Levi, V., and Wehr, T. (1993). Size-based separations of native proteins and SDS-protein complexes using nongel sieving capillary electrophoresis. Amer. Biotechnol. Lab., January, pp. 26-28. 

Flow field flow Cross-flow-based Size-based separation Sample dilution during... 

The size-based selective separation has been in use in the form of dialysis for a long time. The nanofiltration membranes demonstrated here combine both size and chemical transport selectivity and are selective for the particular separation involved. The tubules are prepared as bottleneck tubules as shown in Figure 20.5. The diameter of the tubule may be manipulated, depending on the preparation conditions, resulting in applications based on size-based separations, pH switch-able ion-transport selectivity, manipulation of potential dependent fluxes etc. [75, 95, 96]. 

Fig. 4 Illustration of an SEC calibration curve. High-MW molecules that have K = 0 and low-MW molecules that have K — pass through the column with no separation. For molecules that have 0 < If < 1, size-based separation occurs.

Size-based separations of homogeneous polyelectrolytes, such as DNA, are not possible in free solution electrophoresis [159]. This is due to the proportionality of the friction hydrodynamic force and total charge of the molecule to its length. The friction hydrodynamic forces exerted on the free-drained polymer coil while it moves as well as the accelerating electrostatic force both increase proportionally with the addition of a nucleotide to the chain. This is why one must typically use a sieving media, such as a gel or an entangled polymer solution, to obtain size-based separations of DNA using electrophoresis. 

Chundawat, S. P. S., Venkatesh, B., Dale, B. E. (2007). Effect of particle size based separation of milled com stover on AFEX pretreatment and enzymatic digestibility. Biotechnology and Bioengineering, 96, 219-231. 

Bullock et al. characterized PEGylated superoxide dismutase (SOD) by CZE using a low-pH separation buffer (pH 2.05) [133]. At low pH, protein molecules are completely protonated and the charge differences between individual PEG-mers (i.e., mono-, di-, or tri-PEGmers) are minimized due to neutralization of lysine residues modified by PEG attachment. This results in a predominantly size-based separation of PEGylated proteins. Under low pH conditions, PEG-SOD conjugates were size-dependently separated and the separation pattern was very similar to the MALDI-TOF MS spectrum. 

Therefore, unlike DNA gel electrophoresis, two kinds of protein gel electrophoresis are possible native gel electrophoresis, where the net charge and shape of the protein contribute to the migration properties of the molecules, and denaturing gel electrophoresis, where all the secondary interactions have been disrupted and an overall negative charge has been introduced to the molecule, thereby allowing for a size-based separation. The latter is commonly known as an SDS-PAGE (sodium... 

Figure 1. Sepaiatioii of protein by one and two imensional electrophoresis. A sample of mouse urine was separated by SDS-PAGE (size-based separation), isoelectric focusing (charge based separation) and by a two dimensional (charge, then size) separation. Proteins were visualised by staining with Coomassie blue protein stain or silver.

Both gel filtration chromatography and gel electrophoresis represent the current standard for size-based separation of biomolecules in... 

Size-based separation capability of an inertial microfluidic device can also be characterized using microparticles. Similar to the visualization of particle focusing, pPSV can also be used to observe particle separation. To differentiate the particles of different sizes under microscope, particles with different fluorescent labels are combined with corresponding filter cubes. For example, a mixture of TRITC-labeled 15 pm-diameter particles and FTTC-labeled 20 pm-diameter particles is driven through a vortex-aided device for separation. To visualize the separation at the chamber area, images are... 

Striemer CC et al (2007) Charge- and size-based separation of macromolecules using ultrathin silicon membranes. Natiffe 445 749-753... 

Fig. 3 Application of rDEP to size-based separation of 3 pm and 1 pm particles in a microfluidic reservoir snapshot image (lef() and superimposed image (right). The applied electric field is 5 kV/m DC plus 95 kV/m AC with an AC to DC field ratio, a = 19. The block arrow indicates the particle moving direction (The images are adapted from [2] with permission)...

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