Electrophoresis starch gel

The procedure was developed by Smithies [12] and represents the first application of an electrophoretic procedure in combination with a sieving effect. The apparatus for this purpose is very simple. It consists of two buffer vessels with platinum electrodes and a gel tray placed between them. The contact of the gel layer with electrode buffers is ensured by wicks of thick chromatographic paper. The gel tray is composed of a plastic base (about 10 x 15 cm) and two identical frames placed on top of each other. The whole system is held together by rubber bands. There are several commercially available instruments of this type (e.g.. Desaga Disaphor, Savant Model HGE 312) all provided with a cooling system. 

The applicability of starch gel electrophoresis is today rather limited (for review see Ref. 93) as it has been superseded by a more efficient method polyacrylamide gel electrophoresis. However, there are areas of appUcation such as typing of haemoglobin variants, where starch gel electrophoresis still plays its irreplaceable role. 

Gel layers are prepared from hydrolysed starch for electrophoresis (Sigma St. Louis, MO, Serva Feinbiochemica, Heidelberg, FRG) by suspending in the gel buffer, degassing the mixture and heating it to the moment when viscosity drops 

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Cathepsin D (from bovine spleen) [9025-26-7] Mr 56,000, [EC 3.4.23.5]. Purified on a CM column after ammonium sulfate fractionation and dialysis, then starch-gel electrophoresis and by ullracentrifugal analysis. Finally chromatographed on a DEAE column [Press et al. Biochem J 74 501 I960],... 

Ferguson, KA, Starch-Gel Electrophoresis— Application to the Classification of Pituitary Proteins and Polypeptides, Metabohsm 13, 985, 1964. 

Fig ure 3. Starch gel electrophoresis of hemoglobins. Tris-EDTA-boric acid buffer, pH 9.0. O-Dianisidine stain. 

Att eZcven y-cJuiin voA nts, discovered thus far, exhibit a change In electrophoretic mobility, and starch gel electrophoresis Is the recommended method for their detection. Quantitation of the variant can best be done by chromatography on columns of either DEAE-Sephadex or CM-Cellulose. The quantities of some variants In heterozygotes differ greatly. For Instance, the relative amount (expressed In %F /Fxotal) varies from 20-25% (F-Malta-I) to 10-15% (most Y C >aln variants) to 5-6%... 

The, chain voAiantS are characterized by the presence of two abnormal components, an abnormal Hb-F (02 /2) and an abnormal Hb-A (tt2 32) Of these two, the 02 2 component dominates and the 02 32 component Is often difficult to detect. The methods of choice are starch gel electrophoresis and anion-exchange chromatography using DEAE-Sephadex or DE-52 Cellulose. Chain analyses of these Isolated hemoglobin components will lead to a definitive Identification. 

The defnon6ttLOtion 0 -chain vaAijant6 In heterozygotes Is complicated by the presence of the large amount of Hb-F. Another obstacle Is the nearly Identical electrophoretic mobilities of Hb-A and the minor Hb-Fi component. Despite these difficulties, abnormalities such as AS, SS, AC, CC, SC, and others can readily be detected using cellulose acetate electrophoresis, starch gel electrophoresis, acid agar electrophoresis, and by CM-Cellulose microchromatography to be described In a separate section. 

Hb-B0Jut 6 OK yif can best be demonstrated by either cellulose acetate or starch gel electrophoresis. The amount of Hb-Bart s can differ from 1% to over 80% dependent on the abnormality Involved. Quantitative determination Is most accurately made by CM-Cellulose or CM-Sephadex chromatography. 

Mlcrochromatographlc analyses were made In Ghana, and starch gel electrophoresis In the USA. 112 samples lost In transport 145 samples not mailed 88 samples not Identifiable. 

The validity of diagnosis by this technique has been examined by comparing more than 2,000 samples by the CM-Cellulose procedure, the original CM-Sephadex procedure, and by starch gel electrophoresis. It Is Interesting that occasionally the AS condition at birth Is not diagnosed by the electrophoretic technique. 

Free a-and 3-chains can be demonstrated In a freshly prepared hemolysate using starch gel electrophoresis at alkaline pH. 

Detection of Met(Ferrl-)Hemoglobins (Hb-M) Detection of these variants can be made by starch gel electrophoresis of the ferrl-derlvatlves of hemoglobins In red cell hemolysate using a phosphate buffer, pH 7 0 (25) However, some methemoglobln variants can be separated from normal Hb-A at pH 9 0 (40) ... 

The presence of Individual chains In a hemoglobin variant can also be demonstrated by electrophoresis at alkaline pH after the protein has been dissociated Into Its subunits through exposure to 6 M urea In the presence of 3-mercaptoethanol. The buffer is either a barbital buffer or a tris-EDTA-boric acid buffer, pH 8.0 - 8.6, and contains 6 M urea and 3-niercapto-ethanol. Dissociation of the hemoglobin Into subunits Is best accomplished In a mixture of 1 ml 10 g% Hb (or whole hemolysate), 4 ml 6 M urea barbital or tris-EDTA-boric acid buffer, and 1 to 1.5 ml 3-mercaptoethanol. After 30 minutes to 1 hour the sample Is subjected to cellulose acetate or starch gel electrophoresis. Each chain has a specific mobility and an alteration In electrophoretic mobility easily Identifies the abnormal chain. 

Fig. 4. — Monitoring of the Multiple Molecular Forms of Tomato Pectinesterase by Starch-gel Electrophoresis.98 [ENZ, detection of pectinesterase activity by paper print with pectin and Bromothymol Blue PROT, protein staining with nigrosin O, origin. Key A, 1 crude tomato extract after ammonium sulfate salting-out, and dialysis 2 pectinesterase fraction from column of DEAE-Sephadex A-50 3 and 4 pectinesterase fractions from column of Sephadex G-75. B, Two parts of the same gel after horizontal slicing 1, 500 fig of the isolated form of pectinesterase from a column of CM-Seph-adex C-50 with 175 mM phosphate-sodium chloride buffer 2, active fraction at 150 mM buffer 4 and 5, 250 fig and 1 mg of the isolated form of pectinesterase, respectively.]...

By use of starch-gel electrophoresis, the total extract of bananas, and the fractions obtained after separation on DEAE-Sephadex A-50, were found to contain six multiple forms of pectinesterase having electrophoretic patterns different from those of tomato pectinesterase.103... 

The only pectic enzyme thus far obtained in crystalline form is the endo-D-galacturonanase from Acrocylindrium.209 Crystallization of the enzyme from a solution of ammonium sulfate was preceded by chromatography on calcium phosphate, Duolite CS 101, and DEAE-cellulose, and by starch-gel electrophoresis. 

Fig. 1. Schematic presentation of the protein pattern of the common Hp types in pure form after starch-gel electrophoresis. The protein pattern of a normal serum belonging to Hp type 1-1 is given at the bottom. The cathodic part is excluded.

The saturated HpHb complex of type 1-1 was formed in solution and mixed with a pure solution of globin. The proteins were then separated by starch gel electrophoresis with a neutral phosphate buffer. The procedure was afterward repeated, but with a solution of Hp-globin complex mixed with an Hb solution. 

Smithies vertical starch gel electrophoresis (S7) separates the plasma proteins more distinctly than any other method. If the Hp concentration is normal, the Hp type can generally be recognized directly after the staining for proteins, but sensitive and more specific staining for heme groups, e.g., benzidine, o-dianisidine (04), and malachite green (N5) are preferable. This technique consumes more hydrolyzed starch than the simpler original horizontal electrophoresis technique (S5). 

The Hb solutions generally used are obtained by simple osmotic hemolysis of normal red cells followed by elimination of the ghosts. The molecular heterogeneity of such solutions of adult Hb is revealed by the starch-gel electrophoresis. The Hb line is therefore not quite distinct, which is a minor drawback when the solutions are used for Hp typing. To stabilize the Hb solutions, it is advisable to bubble CO through them before they are ampouled and stored in the frozen state. 

The starch is boiled with 0.007 M phosphate buffer, pH 7.5, the electrode vessels are filled with 0.03 M phosphate buffer of pH 7.0, which is also used for the bridges. Smithies simple plastic trays (S5) for horizontal starch gel electrophoresis will give results clear enough for routine work. 

Figure 3.25 Starch gel electrophoresis of human serum proteins. Samples 1 and 2 are normal while samples 3, 4 and 5 show an extra band adjacent to the normal albumin which is due to the presence of bis-albumin. Sample 7 contains a myeloma protein that has remained near the origin. (Photograph by permission of Dr D. Brocklehurst, Department of Clinical Chemistry, Doncaster Royal Infirmary, UK.)...

In the 1950s, new methods of protein separation were developed that enabled the systematic study of molecular variation in many more human proteins. Starch gel electrophoresis allowed the separation of closely related protein variants by differences in charge and molecular size. Smithies (1955) detected the amazing polymorphism of haptoglobin. In later years the method was extended to the study of allozymes (enzyme polymorphisms). 

Smithies, O. (1962). Starch gel electrophoresis. Archiv. Biochem. Biophys. Suppl. 1, 125-131. 

In 1956, Smithies and Poulik first used 2-DE combining paper and starch gel electrophoresis to separate serum proteins. Nearly 20 years later, polyacrylamide was applied as a support medium. Charge-based protein separation followed as isoelectric focusing (IEF), applied to SDS-PAGE. Later, urea and nonionic detergents were used in IEF-2DE. The most significant achievement was the separation of proteins from E. coli. 

A-Acetyl neuraminic acid aldolase [from Clostridium perfringens, A-acetylneuraminic acid pyruvate lyase] [9027-60-5] [EC 4.1.3.3]. Purified by extraction with H20, protamine pptn, (NH4)2S04 pptn, Me2CO pptn, acid treatment at pH 5.7 and pptn at pH 4.5. The equilibrium constant for pyruvate + n-acetyl-D-mannosamine ++ /V-acetylneuraminidate at 37° is 0.64. The Km for A-acetylneuraminic acid is 3.9mM in phosphate at pH 7.2 and 37°. [Comb and Roseman Methods in Enzymology 5 391 1962). The enzyme from Hogg kidney (cortex) has been purified 1700 fold by extraction with H20, protamine sulphate pptn, (NH4)2S04 pptn, heat treatment between 60-80°, a second (NH4)2S04 pptn and starch gel electrophoresis. The Km for A-acetylneuraminic acid is 1.5mM. [Brunetti et al. JBC 237 2447 1962). 

Figure 3.23. Starch-gel electrophoresis of whole milk samples in urea and mercaptoethanol. Letters on the bottom half of the photograph refer to the genetic types of the milk proteins. (Photograph courtesy Dr. W. Michalak, Warsaw, Poland). (From Thompson 1970. Reprinted with permission of the American Dairy Science Association.)...

Beil, K. 1962. One-dimensional starch-gel electrophoresis of bovine skim milk. Nature 195, 705-706. 

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