Positional reproducibility of protein spots in two‐dimensional polyacrylamide gel electrophoresis using immobilised pH gradient isoelectric focusing in the first dimension: An interlaboratory comparison

Corbett, Joseph M.; Dünn, Michael J.; Posch, Anton; Görg, Angelika

doi:10.1002/elps.11501501182

Cited by 151 publications

(71 citation statements)

References 26 publications

(13 reference statements)

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“…The first dimension (IEF) was performed using the Ettan IPGphor IEF unit (Amersham) and 7-cm strips, pH 4 to 7 (14,19). The isoelectric gels were first passively rehydrated in a total volume of 125 l of rehydration solution (8 M urea eIF2␣ dephosphorylation assay.…”

mentioning

confidence: 99%

Control of α Subunit of Eukaryotic Translation Initiation Factor 2 (eIF2α) Phosphorylation by the Human Papillomavirus Type 18 E6 Oncoprotein: Implications for eIF2α-Dependent Gene Expression and Cell Death

Kazemi

Papadopoulou

et al. 2004

Molecular and Cellular Biology

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confidence: 99%

Control of α Subunit of Eukaryotic Translation Initiation Factor 2 (eIF2α) Phosphorylation by the Human Papillomavirus Type 18 E6 Oncoprotein: Implications for eIF2α-Dependent Gene Expression and Cell Death

Kazemi

Papadopoulou

et al. 2004

Molecular and Cellular Biology

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“…The molecular mass and pI of a protein are its distinctive features. Given spots on different 2DE gels that have the same molecular masses and pIs are thought to be the identical protein species, provided that the electrophoretic patterns on the 2DE gels are similar to each other (Bjellqvist et al 1994;Corbett et al 1994;Wang et al 2000). Since the electrophoretic patterns of particulate proteinaceous materials were very similar ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Characterization of particulate proteins in Pacific surface waters

Saijo

Tanoue

2004

Limnology & Oceanography

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We investigated molecular characteristics of particulate proteins in Pacific surface waters using two-dimensional electrophoresis (2DE). Most proteinaceous materials estimated by dye-binding methods were characterized by the 2DE unresolved acidic materials with a broad range of molecular mass and the 2DE unresolved low molecular mass materials with a broad range of isoelectric point. The 2DE unresolved acidic and low molecular mass materials were considered to comprise peptides conjugated with acidic saccharides and degradation products (peptides) of proteins, respectively, which indicates that almost all proteins in living organisms failed to survive in detrital particulate organic matter (POM). Nevertheless, 23 discrete proteins were distinguished by the 2DE. Electrophoretic patterns of the discrete proteins indicated that they were a component of detrital POM. Three discrete proteins were subjected to N terminal amino acid sequence analysis. Two proteins out of three could not be determined because their N termini were blocked, and one protein was determined from the N terminus to the ninth amino acid residue. A homology search revealed that the N terminal amino acid sequence of the protein agreed completely with that of 70 kDa heat shock protein (HSP70) derived from photosynthetic organisms. HSP70 is a major member of the molecular chaperones that protect or repair proteins from damage under conditions of environmental stress. The occurrence of HSP70 in this study demonstrated that phytoplankton were able to induce the molecular chaperone(s). Clarification of factor(s) controlling induction of chaperones will enable us to assess the actual environmental stress on phytoplankton at the biomolecular level.The particulate organic matter (POM) in surface water is a complex mixture of living biomass and nonliving detritus. The contribution of living biomass to the bulk POM is generally 1 order of magnitude lower than that of detrital organic matter (e.g., Volkman and Tanoue 2002). The particulate combined amino acids (PCAA) are the largest identified fraction in surface POM (e.g., Wakeham et al. 1997), and dye-binding colorimetric methods demonstrated that proteinaceous materials were a major component of POM (Setchell 1981;Long and Azam 1996). Phytoplankton is a primary producer of organic matter in the sea, and most combined amino acids in phytoplankton are in the form of proteins. However, heterotrophic processing may convert cellular proteins of phytoplankton to other forms before its incorporation into detrital pool. At present the chemical nature of the detrital combined amino acids and proteinaceous materials is not well documented, and the processes by which organic matter produced by phytoplankton is transferred to detrital POM are not clear. The characterization of proteinaceous materials in POM at the molecular level is 1 Corresponding author (tanoue@ihas.nagoya-u.ac.jp). AcknowledgmentsWe thank the captains and crews of the RV Hakuho-maru KH93-4, KH99-3, RV Tansei-maru KT00-13, and TS Seisui-ma...

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“…Intensity of each spot was quantified by calculation spot volume after normalization of the image using the total spot volume normalization method multiplied by the total area of all the spots. The reproducibility of spot position was calculated with Gorbett's method [7]. Molecular mass weight and pI values were determined according to standard protein markers covering the range of 10-100 KDa and the immobilized pH gradient strips.…”

Section: Methodsmentioning

confidence: 99%

Serum biomarkers analyzed by LC-MS/MS as predictors for short outcome of non-small cell lung cancer patients treated with chemoradiotherapy

Huang¹,

Ding²,

Li³

et al. 2012

neo

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Purpose: To find potential serum biomakers that can predict clinical outcome upon treatment in non-small cell lung cancer (NSCLC) by analyzing differential proteins in serum with different sensitivity of chemoradiotherapy (CRT).Materials and Methods: Sera were collected from 37 NSCLC patients before they were treated with concurrent cisplatinbased CRT. According to the outcome of CRT, the patients were divided into sensitive group and resistant one. The proteins in sera were separated by two-dimensional gel electrophoresis after the high abundance proteins were removed from sera. The significantly differentially expressed proteins between two groups were analyzed by liquid chromatography and tendem mass spectrometry (LC-MS/MS). Then, an additional 50 serum samples were used for ELISA analysis to validate the identified proteins that we got in the experiment.Results: Proteins in sera of each group were successfully separated on 2D gels. There were significant discrepancies in serum protein expression between NSCLC patients with different CRT sensitivity. Among eight differently expressed proteins, six proteins were successfully identified with five of which higher expressed and one lower expressed in resistant group. The increased Alpha-1-antitrypsin (α1-AT) level in resistant group comparing to sensitive one were validated by ELISA analysis (p<0.05).Conclusions: Proteomic approach may serve as a useful method in detecting the potential biomarkers for predicting the outcome of treatment in NSCLC patients. NSCLC patients with high α1-AT level in serum before CRT may have a worse treatment outcome.

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Positional reproducibility of protein spots in two‐dimensional polyacrylamide gel electrophoresis using immobilised pH gradient isoelectric focusing in the first dimension: An interlaboratory comparison

Cited by 151 publications

References 26 publications

Control of α Subunit of Eukaryotic Translation Initiation Factor 2 (eIF2α) Phosphorylation by the Human Papillomavirus Type 18 E6 Oncoprotein: Implications for eIF2α-Dependent Gene Expression and Cell Death

Control of α Subunit of Eukaryotic Translation Initiation Factor 2 (eIF2α) Phosphorylation by the Human Papillomavirus Type 18 E6 Oncoprotein: Implications for eIF2α-Dependent Gene Expression and Cell Death

Characterization of particulate proteins in Pacific surface waters

Serum biomarkers analyzed by LC-MS/MS as predictors for short outcome of non-small cell lung cancer patients treated with chemoradiotherapy

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