Buffer optimization for high resolution of human lung cancer tissue proteins by two-dimensional gel electrophoresis

Lee, Ki-Beom; Pi, KyungBae; Lee, Kee-Man

doi:10.1007/s10529-008-9837-8

Cited by 5 publications

(2 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, in the present study, to avoid an electroendosmosis effect and increase procedure reproducibility, the authors suggest concentrations of about 100–150 mM DTT for tear proteome separation as usually used in many high cysteine content (blood, platelets, etc.) samples . Of course, the exact choice of reductant concentration depends on the situation in the experiment.…”

Section: Resultsmentioning

confidence: 99%

Enhanced reproducibility of the human gel‐based tear proteome maps in the presence of di‐(2‐hydroxyethyl) disulfide

Saraygord‐Afshari

Naderi-Manesh

Naderi

2014

Biotech and App Biochem

View full text Add to dashboard Cite

Patterns obtained in two-dimensional gel electrophoresis (2-DE) in the previously published articles suggest a varying number of proteins. To seek the cause of this variation, we investigated the effect of reduction power on the overall tear proteome maps. To this end, the buffers of two reducing agents, dithiothreitol (DTT) at nine different concentrations and di-(2-hydroxyethyl) disulfide (HED), were examined. The assay showed that HED clearly improved 2-DE resolution, increased the number of detectable protein spots, and offered well-resolved chain regions in comparison with those treated with DTT. Furthermore, this study introduced increasing the reduction power as a remedy to increase the reproducibility of two-dimensional human tear proteome maps. In addition, the results of our assessment showed that improved reduction efficiency was accompanied by increased procedure reproducibility from 42% to 89%.

show abstract

Section: Resultsmentioning

confidence: 99%

Enhanced reproducibility of the human gel‐based tear proteome maps in the presence of di‐(2‐hydroxyethyl) disulfide

Saraygord‐Afshari

Naderi-Manesh

Naderi

2014

Biotech and App Biochem

View full text Add to dashboard Cite

show abstract

“…Conversely, two papers report consistently poor results with the use of a TBP‐containing buffer for the solubilization of human lung cancer samples 56. Specifically, on a membrane‐enriched fraction TBP decreased the yield of hydrophobic proteins but also caused some proteins, such as hsp60, prohibitin and actin, to be resolved as a string of spots.…”

Section: Analysis Of Protein Samples Under Non‐reducing Conditionsmentioning

confidence: 99%

Other than IPG‐DALT: 2‐DE variants

2010

View full text Add to dashboard Cite

Special problems require special solutions. That is why, despite the trend towards standardization of the procedures in proteomic investigations, a number of alternative protocols, based on 2-DE, have been devised with time and are applied to the resolution of proteins on which the performance of IPG-DALT is poor. The difficult-to-handle samples include high pI, high molecular size and high hydrophobicity proteins. The protocol variants entail changes in the gel media and/or the use of unusual additives (often surfactants or solvents). But also: Specific questions require specific answers. When the aim is not the resolution of individual polypeptide chains in a fully unfolded state but the analysis of higher-order structures (proteins assembled from subunits, complexes assembled from interacting proteins) the standard conditions under which IPG-DALT is performed turn inadequate. Electrophoresis is then performed either in first dimension or in both first dimension and second dimension under non-denaturing (native) and/or non-reducing conditions.

show abstract

Proteomic and functional profiles of a follicle‐stimulating hormone positive human nonfunctional pituitary adenoma

et al. 2015

View full text Add to dashboard Cite

Nonfunctional pituitary adenoma (NFPA) is highly heterogeneous with different hormone-expressed subtypes in NFPA tissues including follicle-stimulating hormone (FSH) positive, luteinizing hormone-positive, FSH/luteinizing hormone-positive, and negative types. To analyze in-depth the variations in the proteomes among different NFPA subtypes for our long-term goal to clarify molecular mechanisms of NFPA and to detect tumor biomarker for personalized medicine practice, a reference map of proteome of a human FSH-expressed NFPA tissue was described here. 2DE and PDQuest image analysis were used to array each protein. MALDI-TOF PMF and human Swiss-Prot databases with MASCOT search were used to identify each protein. A good 2DE pattern with high level of between-gel reproducibility was attained with an average positional deviation 1.98 ± 0.75 mm in the IEF direction and 1.62 ± 0.68 mm in the SDS-PAGE direction. Approximately 1200 protein spots were 2DE-detected and 192 redundant proteins that were contained in 141 protein spots were PMF-identified, representing 107 nonredundant proteins. Those proteins were located in cytoplasm, nucleus, plasma membrane, extracellular space, and so on, and those functioned in transmembrane receptor, ion channel, transcription/translation regulator, transporter, enzyme, phosphatase, kinase, and so on. Several important pathway networks were characterized from those identified proteins with DAVID and Ingenuity Pathway Analysis systems, including gluconeogenesis and glycolysis, mitochondrial dysfunction, oxidative stress, cell-cycle alteration, MAPKsignaling system, immune response, TP53-signaling, VEGF-signaling, and inflammation signaling pathways. Those resulting data contribute to a functional profile of the proteome of a human FSH-positive NFPA tissue, and will serve as a reference for the heterogeneity analysis of NFPA proteomes.

show abstract

Buffer optimization for high resolution of human lung cancer tissue proteins by two-dimensional gel electrophoresis

Cited by 5 publications

References 17 publications

Enhanced reproducibility of the human gel‐based tear proteome maps in the presence of di‐(2‐hydroxyethyl) disulfide

Enhanced reproducibility of the human gel‐based tear proteome maps in the presence of di‐(2‐hydroxyethyl) disulfide

Other than IPG‐DALT: 2‐DE variants

Proteomic and functional profiles of a follicle‐stimulating hormone positive human nonfunctional pituitary adenoma

Contact Info

Product

Resources

About