Separation gel

Another difference between other types of electrophoresis and disc electrophoresis is that the molecules in a sample do not start to significantly separate until entering the separating gel. A discontinuous gel system may be used with almost any type of 2one electrophoresis appHcation. 

Size exclusion was first noted in the late fifties when separations of proteins on columns packed with swollen maize starch were observed (Lindqvist and Storgards, 1955 Lathe and Ruthven, 1956). The run time was typically 48 hr. With the advent of a commercial material for size separation of molecules, a gel of cross-linked dextran, researchers were given a purposely made material for size exclusion, or gel filtration, of solutes as described in the classical work by Porath and Flodin (1959). The material, named Sephadex, was made available commercially by Pharmacia in 1959. This promoted a rapid development of the technique and it was soon applied to the separation of proteins and aqueous polymers. The work by Porath and Flodin promoted Moore (1964) to apply the technique to size separation, gel permeation chromatography of organic molecules on gels of lightly cross-linked polystyrene (i.e., Styragel). 

Fig. 5. Thirty-four out of 35 potential apo(a) isoforms, separated by SDS-agarose gel electrophoresis. The photograph represents a composite of two separate gels with the reference mixture (St) and 17 different samples applied to each gel. Samples were selected to represent each of the observed isoforms. Twenty-nine samples had single-handed patterns and five samples had double-banded patterns. The double banded types and every fifth single-handed phenotype are indicated at the bottom of each gel lane. [With permission of Marcovina et al. (M12).]...

Separation of proteins. Aliquots of biological fluids (blood serum or allantoic fluid) were mixed with the Laemmli buffer for the samples. For proteins denaturation samples were boiled for 5 min. Samples were loaded in and separated in the gel by Miniprotean II unit (BioRad). The voltage was 80 V when samples were in concentrating gel and 100 V when in separating gel. The movement of the proteins was tracked by bromophenol blue. 

The Laemmli SDS-PAGE protocol is one of the most important analytical techniques in analytical protein separation. It is a system with discontinuous pH gradient (disc electrophoresis) and consists of a stacking and a separation gel different in acrylamide concentration and pH. The separation gel may be formed with homogenous acrylamide concentration or with an increasing gradient. 

To allow a complete polymerization the separation gel should be made the day before use. To get a smooth surface and to avoid drying the gel, cover the liquid polymerization mixture with a layer of ddH20,1 20 diluted buffer B, or n-butanol. When water or buffer is used, add these liquids at both sides of the sandwich and allow to come together slowly in the middle. After completing the polymerization reaction, a sharp borderUne appears between polyacrylamide gel and liquid. 

The stacking gel is made just before performing the electrophoresis. The residual liquid on top on the separation gel is removed completely using small pieces of filter paper. Then the stacking gel mixture is added and the slot former ( comb ) is in-... 

The applied sample volume depends on the slot size and should be as low as possible, and the concentration should be as high as possible. As an example, with 1 mm gel thickness and 10 to 15 cm separation gel length, the amount of protein should not exceed 20 - 30 pg/mm slot bottom area. 

Start the electrophoresis immediately when all samples, marker protein mixtures, or references are applied, because molecules diffuse through the soft stacking gel and the pH jump between stacking gel and separation gel, which is important for separation power, drops down. 

As a general rule, a voltage of 30-40 volts is useful for introducing the sample into the stacking gel and field strength of 10-15 V/cm separation gel length for the separation is sufficient. If an efficient cooled device is used, the field strength maybe increased. 

Prepare the separation gel and the stacking gel according to Table 2.6. The separation gel may be poured separately and overlaid with ddH20, but the stacking gel also can be carefully poured directly onto the fresh (liquid) separation gel. 

Solutions are gently mixed according to Table 2.9 in the indicated order and filled up with ddH20. Start the polymerization by addition of Soln. F and pour the mixture immediately into the gel cassette. When the appropriate height is reached, cover the liquid with water or n-butanol to get a smooth surface. Prepare the stacking gel as short as possible before starting the electrophoresis to avoid a decrease of the pH jump between stacking and separation gel by diffusion. 

The discontinuous Laemmli SDS-PAGE with polyacrylamide concentration gradient in the separating gel is mosdy used for second dimension in 2D-PAGE (cf Protocol 2.1.1). Of course, other electrophoretic systems are also applicable. 

Prepare the gel as described in Protocol 2.1.1, i.e., first a separation gel followed by a stacking gel without sample application slots. 

Run the electrophoresis as described in Protocol 2.1.1. Cool the gel to about 15 °C during the run. When the tracking dye bromophenol blue has reached the interphase between stacking and separation gel, stop electrophoresis. Remove the IPG strip and move the electrode paper strip to that place where the IPG strip... 

The transfer protocols are the same for DNA and RNA. In opposition to the methods given in Protocol 2.5.3, the forces driving the biomolecules from the separating gel to the receiving membrane are diffusion and capillary flow. This type of transfer is also applicable for proteins, but because the pores of polyacrylamide gels used for protein separation are mostly smaller, transfer times are longer and transfer efficiencies are lower than by electrotransfer. 

Mix the protein dissolved in binding buffer with equilibrated lEC gel. Agitate for several minutes and separate gel and solution either by centrifugation or filtration. Wash and elute the gel on a funnel or in a column. Since the equilibrium is established only once, a nearly complete absorption occurs if the gel is in a large excess. The advantage of batch loading is the easy separation of gel and liquid. 

SDS-polyacrylamide gel electrophoresis Protein samples were analyzed under denaturing conditions in a discontinuous gel system as described by Laemmli [13] using a 5% stacking gel and a 12% separating gel in a vertical gel system (BioRad, Mini Protean 11, CA, USA). 

To saturate serum transferrin with iron, 10 pi of serum and 10 pi 200 pM Fe(III)-citrate are added to 30 pi of double-distilled water. After incubation for 10 min at room temperature the mixture is diluted 1 50 with double-distilled water. A 1 -pi aliquot is loaded onto the PhastGel sample applicator 8/1 and IEF is performed as described above. After separation, gels are incubated with rabbit anti-human transferrin antibody (dilution 1 3 in 150 mM NaCl) for 40 min followed by washing in... 

Acrylamide (20%) with 0.5% bisacrylamide is appropriate for the separating gel. PVDF blots are stained with amido black, and all clearly visible bands are excised for direct microsequencing on the membrane, using an Applied Biosystems 476A or 494 sequencer (37)... 

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