Erythrocytes electrophoresis

Electrophoresis involves the movement of a charged particle through a liquid under the influence of an applied potential difference. A sample is placed in an electrophoresis cell, usually a horizontal tube of circular cross section, fitted with two electrodes. When a known potential is applied across the electrodes, the particles migrate to the oppositely charged electrode. The direct current voltage applied needs to be adjusted to obtain a particle velocity that is neither too fast nor too slow to allow for errors in measurement and Brownian motion, respectively. It is also important that the measurement is taken reasonably quickly in order to avoid sedimentation in the cell. Prior to each measurement, the apparatus should be calibrated with particles of known zeta potential, such as rabbit erythrocytes. 

Fig. 10. A. Acetic acid-urea-triton-X-100 polyacrylamide gel electrophoresis [15] of the histones used to reconstitute 208-12 nucleosome arrays consisting of recombinant H2A.Z (lane 2) or recombinant H2A.1 (lane 3). Lanes 1 and 4 respectively are chicken erythrocyte and calf thymus histones used as markers [42]. B. Ionic strength (NaCl concentration) dependence of the average sedimentation coelRcient (s2o,w) of reconstituted 208-12 nucleosome arrays containing either H2A.1 (O) or H2A.Z ( ) [42]. The dotted line represents the behavior of a 208-12 complex reconstituted with chicken erythrocyte histones [406]. [Reproduced from Abbott D.W. et al. (2001) I. Biol. Chem. 276, 41945-41949, with permission from The American Society for Biochemistry and Molecular Biology.]...

Clearly a number of differences could explain these divergent results. Boiling could cause loss of antigenicity but retention of activity in stimulating the formation of O . Alternately, the Cab might be attached to agarose beads and to erythrocytes in different ways so that different parts of the molecule were exposed to the PMNs. The assays used to detect contamination of Cab by IgG may also differ in sensitivity (immunofluorescence vs. gel electrophoresis of isolated Ca). 

Figure 8-14 SDS-polyacrylamide gel electrophoresis of human erythrocyte ghosts. (A) From untreated cells. (B) From cells digested externally with chymotrypsin. (C) Inside-out vesicles prepared from cells pretreated with chymotrypsin. (D) The same inside-out vesicles after further treatment with chymotrypsin. (E) Polypeptides released hy the chymotryptic treatment of the inside-out vesides. The peptides are numbered according to the system of Steck232 Hb, hemoglobin. From Luna et al233...

One approach to this problem would be to isolate the intact lectin-receptor molecules from the cell membranes and characterize them. Work in this direction has been initiated in our laboratory, and a method for the isolation of the peanut agglutinin receptor from membranes of neuraminidase-treated human erythrocytes on a column of peanut agglutinin-polyacryl-hydrazido-Sepharose has been developed (21). The amino acid composition, D-glucosamine ancHg-galac-tosamine content, and the electrophoretic mobility on polyacrylamide gel electrophoresis in sodium... 

Another isoenzyme with substantial interest is erythrocytic acid phosphatase (EAP) (8, 9, 10). This system has three autosomal allelic genes termed A, B and C. These can be homozygous or heterozygous giving rise to BA, CA and CB phenotypes. Each of these phenotypes is easily distinguished using starch gel electrophoresis with very useful population frequencies of approximately A - 13%, B - 35%, C - 0.2%, BA - 43%, CA - 3%,... 

Figure 2. Erythrocytic acid phosphatase schematic. A schematic drawing illustrating typical results of an EAP determination in a 13%, 1mm starch gel prepared in 0.24M Nall TO, 0.15M trisodium citrate tannic acid buffer diluted 1 100. The electrophoresis is carried out for 41/2 hr at approximately 410 V. The gels are stained by the fluorescence produced after enzymatic hydrolysis by methylumbellifertjl phosphate at 37° C for 1V2 hr.

Human erythrocyte acid phosphatase (EAP) polymorphism was first described by Hopkinson, Spencer and Harris (1). EAP can be classified by electrophoresis into six different phenotypes,... 

Fig. 5.6. Two-dimensional gel electrophoresis of reversibly crosslinked human erythrocyte membranes. Ghosts were crosslinked with methyl 3-(4-azidophenyldithio)propionimidate and electrophoresed on a 0.5% agarose-1.75% polyacrylamide tube gel. The gel was treated with P-mercaptoethanol (10 mM), and electrophoresed in the second dimension on a 4 to 12 % gradient polyacrylamide slab gel. Some of the crosslinked components (mostly oligomers of spectrin) were incompletely cleaved. (Ji et al., 1980).

Figure 7.5 Electrophoretic patterns of erythrocyte lysates bearing abnormal hemoglobins. C, A2, S, F, Al7 Barts, and H are hemoglobins C, A2, S (sickle), Aa (normal), Barts and H, respectively. Electrophoresis was done at pH 8.6, the medium was stained with a protein-specific dye, and the bound dye was quantitated densitometrically. Absorbance is on the y axis, distance moved, on the x axis, Shaded areas represent abnormal patterns. (From a circular by Gelman Instrument Co., Ann Arbor, MI, by permission of the copyright holder.)...

Fibroblasts were selected because they are readily cultivated and radio-labeled and are available from skin explants of a variety of species. Autoradiograms of radiolabeled cellular proteins are more diverse and easily analyzable for molecular systematics than alternatives such as silver-stained serum or erythrocyte protein patterns. We have achieved nearly 100% success rates at minimal discomfort and risk to human subjects by establishing cultures from 3 mm punch biopsies from the upper buttock. Local lidocaine anesthesia is used. Samples are collected following informed consent and under an approved human research protocol. Other species are sampled by dart gun8 or while sedated. Skin samples can be collected from various body sites without compromising the 2D electrophoresis metric, which does not rely on quantitative differences in protein expression. To comply with the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), tissues collected from wild and captive exotic animals must be obtained under specific permits issued by the U.S. Fish and Wildlife Service. 

Low, TY, Seow, TK. and Chung, M.C. (2002) Separation of human erythrocyte membrane associated proteins with one-dimensional and two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Proteomics 2,... 

Rabilloud, X, Blisnick, T., Heller, M., Luche, S., Aebersold, R., Limardi, J. and Braun-Breton, C. (1999) Analysis of membrane proteins by two-dimensional electrophoresis comparison of the proteins extracted from normal or Plasmodium falciparam-infected erythrocyte ghosts. Electrophoresis 20, 3603-3610. 

Figure 2. SDS gel electrophoresis of the products of partial cystine cleavage for several test proteins. A. molecular weight standards, B. yeast alcohol dehydrogenase. C. P-lactoglobulin, D. hen egg lysozyme, E. ovalbumin, F. calf fetal serum fetuin. Molecular weight standards are indicated by arrows on the left side of the gel and are bovine serum albumin (66,300), bovine liver glutamate dehydrogenase (55,400), porcine muscle lactate ddiydiogenase (36,500), bovine erythrocyte carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500), hen egg lysozyme (14,400), bovine lung aprotinin (6,000), unresolved bovine pancreatic insulin A and B chains.

The protein components of a membrane can be readily visualized by SDS-polyacrylamide gel electrophoresis. As discussed earlier (Section 4.1.4). the electrophoretic mobility of many proteins in SDS-containing gels depends on the mass rather than on the net charge of the protein. The gel-electrophoresis patterns of three membranes—the plasma membrane of erythrocytes, the photoreceptor membrane of retinal rod cells, and the sarcoplasmic reticulum membrane of muscle—are shown in Figure 12.16. It is evident that each of these three membranes contains many proteins but has a distinct protein composition. In general, membranes performing different functions contain different repertoires of proteins. 

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