Analysis of Glycoproteins Separated by Two-Dimensional Gel Electrophoresis Using Lectin Blotting Revealed by Chemiluminescence

Gravel, Patricia; Golaz, Olivier; Walzer, Claude; Hochstrasser, Denis F.; Türler, Hans; Balant, L

doi:10.1006/abio.1994.1380

Cited by 36 publications

(16 citation statements)

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“…For lectin blotting, nitrocellulose sheets were blocked overnight with 0.5% (v/v) Tween 20 in PBS. Incubation with peroxidase-conjugated WGA and UEA-1, was carried out for 3 h at room temperature (Gravel et al, 1994). Lectin stock solutions containing 1 mg/ml protein were diluted 1:500 and 1:1000 for UEA-1 and WGA staining, respectively.…”

Section: Lectin Blotting and Immunoblot Analysismentioning

confidence: 99%

Lectin-based proteomic profiling of aged skeletal muscle: Decreased pyruvate kinase isozyme M1 exhibits drastically increased levels of N-glycosylation

O’Connell

Doran

Gannon

et al. 2008

European Journal of Cell Biology

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Since various neuromuscular diseases are associated with abnormal glycosylation, it was of interest to determine whether this key post-translational modification is also altered in aged skeletal muscle. Lectins represent highly versatile carbohydrate-binding proteins that are routinely employed for the characterization of glycoproteins. Here, we used the lectin wheat germ agglutinin (WGA) for the proteomic profiling of senescent fibers. WGA labeling of the soluble proteome from 3-month-versus 30-month-old rat gastrocnemius muscle, following two-dimensional gel electrophoretic separation, resulted in the identification of 13 distinct protein species. Analysis of WGA binding levels, in conjunction with mass spectrometric fingerprinting, revealed that one isoform of a major metabolic muscle protein exhibited a drastic alteration in the content of sialic acid and N-acetylglucosaminyl sugar residues. Pyruvate kinase isoform M1 with protein accession number gi|16757994|, exhibiting a pI of 6.6 and an apparent molecular mass of 57.8 kDa, showed a six fold increase in N-glycosylation and a three fold decrease in protein expression. In contrast to comparable levels of N-glycosylated proteins in young adult versus senescent muscle, as judged by fluoresceinconjugated WGA labeling of transverse muscle cryosections, staining with antibodies to the M1 isoform of pyruvate kinase showed reduced expression of this cytosolic element. Furthermore, activity assays demonstrated a reduced activity of this glycolytic enzyme in senescent muscle. This agrees with the idea that abnormal post-translational modifications in key metabolic enzymes may be involved in the conversion of aged muscle to slower twitch patterns and a drastic shift to more aerobic-oxidative metabolism.

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Section: Lectin Blotting and Immunoblot Analysismentioning

confidence: 99%

Lectin-based proteomic profiling of aged skeletal muscle: Decreased pyruvate kinase isozyme M1 exhibits drastically increased levels of N-glycosylation

O’Connell

Doran

Gannon

et al. 2008

European Journal of Cell Biology

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“…15,16 IEF was performed with a Multiphor™ II apparatus (LKB, Bromma, Sweden) connected to a Model 3000 Xi power supply (Bio-Rad, Hercules, CA). Then, 300 V tension was applied for 8 h and then increased to 5000 V for 17 h. The temperature was maintained at 10°C.…”

Section: -D Pagementioning

confidence: 99%

Kinetics of blood component adsorption on poly(D,L-lactic acid) nanoparticles: Evidence of complement C3 component involvement

Allémann

Gravel

et al. 1997

J. Biomed. Mater. Res.

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After intravenous administration, nanoparticles suffer a major drawback in that they are rapidly and massively taken up by the cells of the mononuclear phagocyte system. The mechanisms involved in the opsonization, adhesion, and internalization of biodegradable nanoparticles by the mononuclear phagocyte system are still poorly understood. In this work, the kinetics of blood protein adsorption onto nanoparticles of poly(D,L-lactic acid) prepared by the salting-out technique was investigated. Nanoparticles of 312 nm were incubated for variable periods of time (5-60 min) in human serum and citrated plasma. After incubation, the particles were washed and the proteins detached from them, denatured, and analyzed by two-dimensional polyacrylamide gel electrophoresis. In plasma, the predominant protein was immunoglobulin G (IgG), and the amount adsorbed was not dependent on incubation time. Albumin amounts were high for short incubation periods but decreased as a function of time, whereas apolipoprotein E levels increased significantly as a function of the incubation period. Owing to the possible complement cascade inactivation by addition of citrate to plasma, the kinetics of adsorption was also evaluated in serum. In this medium, adsorption of complement C3 components onto the surface of the nanoparticles was clearly evidenced by spots of increasing intensity and area, reaching levels comparable to those of the omnipresent IgG. This result confirms the important role of complement components in the opsonization process of poly(D,L-lactic acid) particles.

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“…(ii) The second method of identification was immunoblotting using the sensitive chemiluminescence detection method [22]. Four cytoskeletal-related proteins were identified: a-actinin, gelsolin, vinculin and tropomyosin.…”

Section: Identification Of Cytosol Platelet Proteinsmentioning

confidence: 99%

“…The temperature was maintained at 10°C. The IPG strips were then equilibrated as described previously [22,231. After equilibration, the IPG strip gel was transferred to a 9-16010 gradient polyacrylamide gel [23,24].…”

Section: -D Pagementioning

confidence: 99%

Human blood platelet protein map established by two‐dimensional polyacrylamide gel electrophoresis

Gravel¹,

Sanchez

Walzer³

et al. 1995

Electrophoresis

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Two-dimensional (2-D) maps of cytosol and enriched-membrane platelet proteins has allowed the identification of more than 25 spots by three different methods: matching of the platelet gels with other 2-D reference maps, immunoblotting with chemiluminescence detection, and N-terminal sequencing. Different G protein (guanosine triphosphate-binding protein) subunits, cytoskeletal proteins, and proteins common to the human liver, red blood cells and plasma were identified. The two platelet protein maps presented here contribute to the project of identification of human cell and body fluid proteins. They may serve as working tools since platelets are popular models for the study of central nervous system neurotransmitter systems and stimulus-response coupling mechanisms.

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Analysis of Glycoproteins Separated by Two-Dimensional Gel Electrophoresis Using Lectin Blotting Revealed by Chemiluminescence

Cited by 36 publications

References 0 publications

Lectin-based proteomic profiling of aged skeletal muscle: Decreased pyruvate kinase isozyme M1 exhibits drastically increased levels of N-glycosylation

Lectin-based proteomic profiling of aged skeletal muscle: Decreased pyruvate kinase isozyme M1 exhibits drastically increased levels of N-glycosylation

Kinetics of blood component adsorption on poly(D,L-lactic acid) nanoparticles: Evidence of complement C3 component involvement

Human blood platelet protein map established by two‐dimensional polyacrylamide gel electrophoresis

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