Efficient method for vsualization and isolation of proteins resolved in polyacrylamide gels

Francis, Ralph T.; Davie, James R.; Sayre, Mike; Rocha, E; Ziemer, Forest; Riedel, Georgia

doi:10.1016/s0021-9673(01)92699-8

Cited by 10 publications

(6 citation statements)

References 10 publications

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“…The pooled fractions were dialyzed against 0.1 N acetic acid, followed by water, and lyophilized. Trout histone H2A.Z was isolated from fraction 3 (see Figure 2) by preparative AUT-polyacrylamide gel electrophoresis (Francis et al, 1984).…”

Section: Methodsmentioning

confidence: 99%

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Changes in the histone H2A variant H2A.Z and polyubiquitinated histone species in developing trout testis

Sy²,

Rg³

et al. 1987

Biochemistry

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The trout histone H2A variant H2A.Z has been identified by its electrophoretic mobility on two-dimensional polyacrylamide gels and its N-terminal amino acid sequence. Similar to bovine H2A.Z and chicken H2A.F (also called H2A.Z and M1), the trout H2A.Z had a two-residue extension when aligned with trout H2A and a 67% sequence homology with the N-terminal portion of trout H2A. The first 29 amino acids of trout H2A.Z were identical with those of chicken H2A.F and differed from those of bovine H2A.Z at only one position. Thus, the N-terminal part of histone H2A.Z appears to be highly conserved. The levels of histone H2A.Z and ubiquitinated species of the histones H2A, H2A.Z, and H2B, which were detected with an anti-ubiquitin antibody, were studied at various stages of trout testis development. At the final stages of spermatogenesis in trout, histones are replaced by protamines. Ubiquitinated and diubiquitinated histone H2A remained at similar levels in early and late stage testis nucleohistone. In the late stage testis chromatin (nucleohistone), ubiquitinated histone H2A.Z was not detected, the level of ubiquitinated histone H2B was reduced, and the amount of diubiquitinated histone H2B increased. There was also a marked reduction in the level of histone H2A.Z. This observation suggests nucleosomes with this histone variant were selectively disassembled during the transition from nucleohistone to nucleoprotamine, indicating that protamine deposition is not a random process in rainbow trout.

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Section: Methodsmentioning

confidence: 99%

“…Histones were electrophoretically resolved on a 0.5 mm thick AUT gel. Following electrophoresis, the bands were visualized by staining with 8-anilino-lnaphthalenesulfonic acid (Francis et al, 1984). The gel slices containing the protein of interest were placed perpendicular to the surface of the stacking gel.…”

Section: Methodsmentioning

confidence: 99%

Changes in the histone H2A variant H2A.Z and polyubiquitinated histone species in developing trout testis

Sy²,

Rg³

et al. 1987

Biochemistry

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“…The protein was recovered from each slide by allowing it to diffuse into 2 ml of a 50 mM ammonium bicarbonate solution in a polypropylene tube over a period of 12 h at 4 °C with gentle agitation. Electroelution was carried out in a tube gel apparatus against a 3,500-dalton-cutoff dialysis membrane, as described by Francis et al (12). The fractions thus obtained were assayed for activity, and subjected to polyacrylamide gel electrophoresis in a denaturing system containing sodium dodecyl sulfate (SDS-PAGE) in 10-15% linear gradient gels under nonreducing and reducing conditions to assess purity.…”

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confidence: 99%

Purification of cachectin, a lipoprotein lipase-suppressing hormone secreted by endotoxin-induced RAW 264.7 cells.

Beutler¹,

Mahoney²,

Trang³

et al. 1985

The Journal of Experimental Medicine

759

246

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Previous studies have indicated that endotoxin and other bacterial and protozoal products can stimulate macrophages to produce a factor that can suppress the activity of the enzyme lipoprotein lipase (LPL), in vivo and in vitro. In the present report we describe the purification of this factor, cachectin, to apparent homogeneity from the conditioned medium of endotoxin-stimulated RAW 264.7 cells. The isolated protein has an isoelectric point of 4.7 and a subunit molecular weight of 17,000. Although cachectin's isoelectric point and molecular weight are similar to those described for interleukin 1, pure cachectin has no leukocyte-activating factor (LAF) activity. Cachectin at a concentration of 10(-11) M has the ability to suppress the LPL activity of the 3T3-L1 adipocyte cell line by 80%. Binding studies using radio-labeled cachectin and 3T3-L1 adipocytes and C2 myotubules revealed approximately 10(4) high-affinity receptors per cell on both cell types (Ka, 3 X 10(9]. Cachectin receptors were also present on liver membranes but were absent on erythrocytes and lymphocytes. The isolation of cachectin and characterization of its receptor should facilitate further investigations into the role of cachectin and other macrophage mediators in the metabolic derangements that occur during infection and cachexia.

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“…The reaction was stopped after 20 h by freezing at -80°C, followed by lyophilization. Digestion products were separated by SDS-PAGE using Tris-borate as running buffer (5). After electrophoresis, the gel was formaldehyde-fixed and stained with Coomassie brilliant blue R-250 (25).…”

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confidence: 99%

Purification and Characterization of Arcelin Seed Protein from Common Bean

Osborn¹,

Burow²,

Bliss³

1988

Plant Physiol.

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Arcelin, a seed protein originally discovered in wild bean accessions, was purified, characterized, and compared to phaseolin, the major seed protein of common bean, and to phytohemagglutinin (PHA), the major bean seed lectin. Arcelin and PHA has several characteristics in common.Both were glycoproteins having similar subunit M, deglycosylated Mn and amino acid compositions. The two proteins were related antigenically and they had the same developmental timing of accumulation. Arcelin also had some hemagglutinating activity, a characteristic associated with lectins. However, several features distinguished arcelin from PHA. Arcelin had a more basic isoelectric point than PHA, greater numbers of basic amino acid residues, additional cysteine residues, and one methionine residue, which PHA lacks. Native PHA protein is a tetramer of subunits, and although a small component of native arcelin protein was also tetrameric, most of the arcelin preparation was dimeric. The hemagglutinating activity ofarcelin was specific only for some pronase-treated erythrocytes. It did not agglutinate native erythrocytes, nor did it bind to thyroglobulin or fetuin aff'mity resins as did PHA. Although arcelin has lectin-like properties, we believe the distinctions between arcelin and PHA warrant the designation of arcelin as a unique bean seed protein.was co-dominant with respect to each other and dominant with respect to alleles for the absence of arcelin. Genes controlling arcelin expression were tightly linked (<0.3% recombination) to those controlling PHA expression. The presence of arcelin in wild bean accessions was correlated with high levels of resistance to two bruchid beetle species (19,24).Although arcelin protein has a different electrophoretic mobility than other major seed proteins, it is not known whether arcelins represent a truly distinct class of seed proteins. The relationship of arcelin to other seed proteins is particularly interesting in light of the tight linkage between arcelin and PHA genes. In this study, we report on the purification and characteriz,tion of the arcelin-1 variant and compare its properties to those of PHA and phaseolin seed proteins. The major seed storage proteins of cultivated common bean (Phaseolus vulgaris) are phaseolin and PHA,2 also referred to as bean lectin. In several wild bean accessions, a third important protein fraction has been observed which had not been described previously in bean cultivars (19,23). This fraction was discovered as a major seed protein having a different electrophoretic mobility than either phaseolin or PHA with which it occurred in seeds of some wild beans. Four electrophoretic variants of this protein were observed which consisted of distinct groups of polypeptides having similar mobilities on two-dimensional gels. This protein was named arcelin and the variants were designated arcelin-1, 2, 3, and 4.There has been only limited characterization of arcelin, but the protein and its genetic control have several interesting features. Arcelin was shown to o...

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Efficient method for vsualization and isolation of proteins resolved in polyacrylamide gels

Cited by 10 publications

References 10 publications

Changes in the histone H2A variant H2A.Z and polyubiquitinated histone species in developing trout testis

Changes in the histone H2A variant H2A.Z and polyubiquitinated histone species in developing trout testis

Purification of cachectin, a lipoprotein lipase-suppressing hormone secreted by endotoxin-induced RAW 264.7 cells.

Purification and Characterization of Arcelin Seed Protein from Common Bean

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