Isoelectric focusing in immobilized pH gradients: Principle, methodology and some applications

Bjellqvist, Bengt; Ek, Kristina; Righetti, Pier Giorgio; Gianazza, Elisabetta; Görg, Angelika; Westermeier, R.; Postel, Wilhelm

doi:10.1016/0165-022x(82)90013-6

Cited by 916 publications

(362 citation statements)

References 30 publications

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“…For each gel, the labeled pool sample and the labeled COD and control samples were mixed and an equal amount of rehydration buffer (2 M thiourea, 7 M urea, 2% dithiothreitol (DTT), 4% CHAPS and 2% pharmalyte (GE HealthCare)) was added and the total volume was brought up to 450 ml by Destreak rehydration buffer (GE HealthCare). 65 For the 'pick gel', 50 mg of pooled sample was labeled with the Cy2 dye, which was combined with 750 mg of unlabeled pool sample. An equal amount of sample buffer was added and the total volume increased to 450 ml by the addition of Destreak rehydration buffer.…”

Section: Cyanine Dye Labelingmentioning

confidence: 99%

“…64,65 Sequential IEF was done by step-and-hold as follows: 10 h at 0 V, 1 h at 100 V, 1 h at 500 V, 1 h at 1000 V, 3 h at 8000 V (gradient; 13.5 kV h), 7.5 h at 8000 V for a total of 75.1 kV h. A maximum current of 50 mA was applied per strip, maintaining the platform temperature at 201C. After IEF, the IPG strips were equilibrated to reduce the disulfide bonds with gentle rocking for 10 min in equilibrating solution (6 M urea, 1.5 M Tris-HCl, pH 8.8, 30% (v/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS) and 2% (w/v) DTT).…”

Section: Two-dimensional Polyacrylamide Gel Electrophoresismentioning

confidence: 99%

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Cytosolic proteomic alterations in the nucleus accumbens of cocaine overdose victims

2006

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Chronic cocaine use in humans and animal models is known to lead to pronounced alterations in neuronal function in the nucleus accumbens (NAc), a brain region associated with drug reinforcement. Two-dimensional gel electrophoresis was used to compare protein alterations in the NAc between cocaine overdose (COD) victims (n = 10) and controls (n = 10). Following image normalization, spots with significantly differential image intensities (P < 0.05) were identified, excised, trypsin digested and analyzed by matrix-assisted laser desorption ionization-time of flight-time of flight. A total of 1407 spots were found to be present in a minimum of five subjects per group and the intensity of 18 spots was found to be differentially abundant between the groups, leading to positive identification of 15 proteins by peptide mass fingerprinting (PMF). Of an additional 37 protein spots that were constitutively expressed, 32 proteins were positively identified by PMF. Increased proteins in COD included b-tubulin, liprin-a3 and neuronal enolase, whereas decreased proteins included parvalbumin, ATP synthase b-chain and peroxiredoxin 2. The present data provide a preliminary protein profile of COD, suggesting the involvement of novel proteins and pathways in the expression of this complex disease. Additional studies are warranted to further characterize alterations in the differentially regulated proteins. Understanding the coordinated involvement of multiple proteins in cocaine abuse provides insight into the molecular basis of the disease and offers new targets for pharmacotherapeutic intervention for drug abuse-related disorders.

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Section: Cyanine Dye Labelingmentioning

confidence: 99%

Section: Two-dimensional Polyacrylamide Gel Electrophoresismentioning

confidence: 99%

Cytosolic proteomic alterations in the nucleus accumbens of cocaine overdose victims

2006

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“…As a possible partial remedy, Tollaksen et al [8] had advocated the use of carbamylated charge standards for testing batches of ampholytes and, in any event, to buy large quantities of ampholytes, possibly from different sources, mix then and store them frozen for years of use! The real solution to this problem came only with the introduction of IPG [9], of course, a technique that, once adopted, eliminated essentially all problems connected with carrier ampholytes. When adopting IPG gels for the first dimension in 2-D maps, Gianazza et al [10] showed, indeed, minute errors in spot position.…”

Section: Introductionmentioning

confidence: 99%

SDS‐PAGE and two‐dimensional maps in a radial gel format

et al. 2010

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A novel method for performing 2-D map analysis is here reported, consisting in a modification of the second dimension run, which is performed not in a conventional square-or rectangular-size gel, but in a radial surface. This has the advantage of permitting resolution of closely adjacent bands, representing strings of isoforms of similar or identical mass but of closely spaced isoelectric points. When used in a monodimensional, SDS-PAGE format, this system allows the simultaneous running of 62 sample tracks. Examples are given of separation of plasma and urinary proteins.

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“…Overall, the use of IPG IEF is one of the highest resolution separation methodologies available for analysis of peptides and proteins [5][6][7][8].…”

Section: Introductionmentioning

confidence: 99%

Calculation of the isoelectric point of tryptic peptides in the pH 3.5–4.5 range based on adjacent amino acid effects

et al. 2008

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Calculation of the isoelectric point of tryptic peptides in the pH 3.5-4.5 range based on adjacent amino acid effects Current algorithms for the calculation of peptide or protein pI, based on the charge associated with individual amino acids, can calculate pI values to within 60.2 pI units. Here, we present a new pI calculation algorithm that takes into account the effect of adjacent amino acids on the pI value. The algorithm accounts for the effect of adjacent amino acids 6 3 residues away from a charged aspartic or glutamic acid, as well as effects on the free C terminus, and applies a correction term to the corresponding pK values. The correction increments are derived from a 5000-peptide training set using a genetic optimization approach. The accuracy of the new pI values obtained with this method approaches the error associated with the manufacture of the IPG strip (,60.03 pI units). The approach is demonstrated for cytosolic cell extracts derived from the breast-cancer cell line DU4475, and from membrane preparations from human lung-tissue samples. One potential application of a more highly accurate pI calculation is data filtering of MS/MS outputs that will allow for more complex database searches including gene finding, and validation, and detection of coding single-nucleotide polymorphisms in their expressed form.

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Isoelectric focusing in immobilized pH gradients: Principle, methodology and some applications

Cited by 916 publications

References 30 publications

Cytosolic proteomic alterations in the nucleus accumbens of cocaine overdose victims

Cytosolic proteomic alterations in the nucleus accumbens of cocaine overdose victims

SDS‐PAGE and two‐dimensional maps in a radial gel format

Calculation of the isoelectric point of tryptic peptides in the pH 3.5–4.5 range based on adjacent amino acid effects

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