Urea gradient gel electrophoresis

The method is less powerful in characterizing proteins lacking tryptophan, for reasons mentioned earlier (see Support Protocol), and does not apply at all to the rare proteins that lack tyrosine as well. Near-UV circular dichroism measurements (unit 7.6) can still be of considerable value for proteins containing tyrosine and, in both cases, both far-UV circular dichroism and urea-gradient gel electrophoresis (unit 7.4) provide powerful contributions to characterization. 

The results obtained from urea gradient gel electrophoresis experiments compare favorably with what has been reported in the literature for the unfolding/folding of carbonic anhydrase. The existence of an intermediate species at 1.2-2M GuHCl or 5M urea has been documented (11,14,16,26,36,37),... 

Figure 1. Urea gradient gel electrophoresis, 0 M urea sample, pH = 7.5, T=18-28 C...

Fig. 3A,B. SDS gradient gel electrophoresis and autoradiography of ADP-ribosylated proteins. Permeabilized HeLa cells (5 X 10 ) were incubated for 20 min at 26°C with 0.133 ijM [ P]-NAD, sp.act. 245.8 Ci mmoP, made up to 1 nM or 100 /lAf with non-radioactive NAD. Each assay contained about 5.9 X 10 cpm. Reactions (75 n ) were stopped by adding 75 n of solution containing 2% (w/v) SDS, 50 mM 2-mercaptoethanol, 6 M urea, 10 mM sodium bisulphite, 5 mM 4-aminobenzamidine, 0.5 mM PMSF, 0.15 iig of pepstatin and 0.15 Mg of leupeptin in 100 mM Tris-HCl pH 5.4 and 40% (v/v) glycerol. The mixture was immediately boiled for 4 min. Slab-gel electrophoresis was carried out in a 6-18% linear gradient polyacrylamide gel made in 25 mM sodium phosphate pH 6.8,1% (w/v) SDS and 3 M urea. A 4% stacking gel was used made in 10 mM sodium phosphate pH 6.0. The electrode buffer was 25 mM sodium phosphate pH 6.8, 1% SDS. Gels were subjected to electrophoresis for 6-7 h at a constant current of 40 mA.

The sample is loaded at a flow-rate of 1 ml/min onto the FPLC column equilibrated with the same MOPS buffer used to resuspend the RNA pellets. The free nucleotides are completely removed with a 5-ml wash with 350 mM NaCl and the RNA is eluted with a 20-ml (350—750 mM NaCl) linear gradient and analyzed by PAGE/urea gel electrophoresis (see later). Up to 2 mg of RNA can be loaded onto and eluted from a 1-ml (of resin) mono Q column without loss of resolution. The homogeneity of RNA in the fractions collected, as seen by gel electrophoresis, should be >90%. The appropriate fractions are pooled and the RNA collected by ethanol precipitation. The RNA pellet is washed twice with 70% ethanol, air-dried, and finally redissolved in DEPC-treated H20. The total recovery after the entire procedure of purification is = 90%. This protocol yields = 800 pmoles of purified 002 mRNA/pmole template DNA. 

Another specialised area of application of continuous zonal electrophoresis is the use of urea gradients to investigate protein stability. The gels for these studies are... 

Recently Altland et al. [116] published a procedure in which polyacrylamide gel electrophoresis is used first followed by isoelectric focusing. Moreover isoelectric focusing in the second dimension is carried out in a gel containing a gradient of from 0 to 8 M urea perpendicular to the pH axis. 

Fig. 3. Preparative and analytical lEC of crude RNA extract. (A) Preparative lEC. Column Nucleogen DEAE-SOO (12x150 mm) sample 30 mg crude RNA extract from viroid-infected plants chromatographic conditions linear gradient from 250 mM to 1 M KCl in 4(X) min 20 mM K-phosphate, pH 6.6, 0.1 mM EDTA, 5 M urea 2 ml/min 21 bar, 22° C. (B) 5% polyacrylamide gel electrophoresis of the peak fractions from (A) as indicated in the chromatogram the unfractionated sample is in slot M. (C) Analytical lEC. Column Nucleogen DEAE-40(X) (3 x50 mm) sample 20 jag of the sample from (A) chromatographic conditions linear gradient from 250 mM to 1 M KCl in 45 min 20 mM K-phosphate, pH 6.6, 0.1 mM EDTA, 5 M urea 1 ml/min 42 bar 24 °C. From (16].

Figure 8. Two-dimensional analysis of proteins in infected HeLa cells. HeLa cells were labelled for 24 hours with (14c) amino acids, washed thoroughly, then infected with poliovirus type 1 Mahoney. Samples were taJcen at sixty min intervals, lysed with 8 M urea, and analyzed by two-dimensional electrophoresis. The first gel was 5% polyacrylamide, 8 M urea, 279 ampholine, pH 5-8, Samples were separated at 500 volts, overnight at 6-7. The gels were sliced, and placed on SHS-polyacrylamide gradient gels, and electrophoresed for 4 5 hours at 20 mA/gel, Gels were stained, dried, and autoradiographed. Gel A uninfected cells Gel B infected with poliovirus for five hours.

Two-dimensionai gel electrophoresis of histones from interphase and mitotic cells treated with dimethyl sulfate. 2-D gel electrophoresis was carried out with proteins separated by nonequilibrium pH gradient electrophoresis in the first dimension and on 8% polyacrylamide slab gels in the second (6, 7). Separation of histones was enhanced by use of a urea solubilization buffer containing protamines to displace histones from DNA (8). The resulting autoradiograms for histones of both nuclei and chromosomes are in Fig. 3. Incubation of mitotic cells with [32P]NAD before chromosomes were isolated led to prominent labeling of histones H2B and H4 (A, B), and spots are also visible at the positions of HIA and HIB. 32p incorporation is also seen at the positions of histones H2A and H3. hi Fig. 3B, a similar pattern is observed for chromosomal histones from cells treated with DMS. The primary acceptors of isotope are H2B and H4, with radioactivity also present at the positions of HIA, HIB, H2A, and H3. A major change in the relative distribution of 32p between species is clearly not a feature of DMS treatment. 

Techniques were developed for preparing relatively large amounts of Chlamydomonas ribosomal subunits on a zonal sucrose density gradient (Fig. 10), for dissociating ribosomal subunits in lithium chloride-urea, and for analyzing ribosomal proteins by polyacrylamide gel electrophoresis in urea or in a two-dimensional urea-SDS system. Using this technique, we found that the 52 S subunit of ery-Mld contains an altered protein (Fig. 

Electrophoretic Methods. Several electrophoretic procedures have been developed to fractionate or purify the various caseins (McKenzie 1971C Thompson 1971 Whitney 1977). Wake and Baldwin (1961) fractionated whole casein by zone electrophoresis on cellulose powder in 7 M urea and 0.02 ionic strength sodium phosphate buffer at pH 7 and 5°C. Payens and co-workers employed several somewhat different electrophoretic conditions for the fractionation and purification of the caseins on cellulose columns (Payens 1961 Schmidt and Payens 1963 Schmidt 1967). Three fractions, as-, k-, and /3-caseins, were separated at pH 7.5 and 30°C with 4.6 M urea-barbiturate buffer. The purification of asi-casein and the separation of the genetic variants of K-casein were accomplished by altering the electrophoretic conditions. Manson (1965) fractionated acid casein on a starch gel column stabilized by a density gradient at 25 °C. 

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