Electrofocusing followed by gradient electrophoresis: A two-dimensional polyacrylamide gel technique for the separation of proteins and its application to the immunoglobulins

Emes, Alan V.; Latner, A. L.; Martin, John Levi

doi:10.1016/0009-8981(75)90146-1

Cited by 29 publications

(3 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The combination of these techniques results in proteins resolved on a gel as spots when stained using silver, fluorescent dyes or Coomassie blue [4]. 2-DE is not a new technique, and indeed a great number of studies have used this technique to assess proteins of interest [5,6]. The application of 2-DE in assessment of clinical samples is comprehensively reviewed by Young and Tracy [7].…”

Section: Proteomic Technologymentioning

confidence: 99%

The use of proteomics for the assessment of clinical samples in research

Aldred

Grant

Griffiths

2004

Clinical Biochemistry

View full text Add to dashboard Cite

Section: Proteomic Technologymentioning

confidence: 99%

The use of proteomics for the assessment of clinical samples in research

Aldred

Grant

Griffiths

2004

Clinical Biochemistry

View full text Add to dashboard Cite

“…Non-denaturing two-dimensional gel electrophoresis (2DE, isoelectric focusing (IEF) of native proteins followed by pore-gradient electrophoresis) was first reported by Emes et al (1975), and then developed by Manabe et al (1979) and Felgenhauar and Hagedorn (1980). Recently, we have reported that proteomic analysis involving enzyme activity is performed using non-denaturing 2DE and mass spectrometry (MS) (Shimazaki and Manabe, 2005;Shimazaki et al, 2003Shimazaki et al, , 2004Shimazaki et al, , 2006.…”

Section: Introductionmentioning

confidence: 99%

Analysis of lipid hydrolytic activity by esterase on blotting membrane followed by separation using non‐denaturing two‐dimensional gel electrophoresis

Shimazaki

Miyamoto

2007

Biotech & Bioengineering

View full text Add to dashboard Cite

After separation by microscale non-denaturing two-dimensional gel electrophoresis (2DE) and transferring to a blotting membrane, major proteins are detected by a staining of direct blue 71 in a neutral solution. The carboxylesterase on the membrane hydrolyzes phosphatidylcholine after the spot of carboxylesterase is excised from the membrane, and incubated with phosphatidylcholine. Lipids of human serum proteins and the purified human high density lipoprotein (HDL) are removed by enzymatic hydrolysis when human serum proteins and the purified HDL are respectively incubated with the spot of carboxylesterase on the membrane. These results indicate that carboxylesterase on the membrane hydrolyzes not only lipids such as phosphatidylcholine but also lipids of lipoproteins such as HDL after separation by the 2DE, transferring to the membrane and staining without impairing the activity. These results also indicate that a micro-immobilized enzyme reactor on the membrane can be produced when biological enzymes are separated by microscale 2DE, transferred to the membrane and stained without impairing their activities.

show abstract

“…1 These classes of lipoproteins consist of particles that are very heterogeneous in size, density and lipid and apolipoprotein composition. This has been con®rmed by several analytical methods, such as sequential and gradient ultracentrifugation, 1 gradient gel electrophoresis, 2 isoelectric focusing, 3 two-dimensional gel electrophoresis 4 and chromatography. 5 Although these methods are very useful and fairly easy for routine clinical diagnostic analysis with relatively high resolution, it is necessary to combine the results obtained by two or more independent methods to obtain more detailed information about a subpopulation of lipoproteins.…”

mentioning

confidence: 97%

Capillary isotachophoretic analysis of serum lipoproteins using a carrier ampholyte as spacer ion

Inano

Tezuka²,

Miida³

et al. 2000

Ann Clin Biochem

View full text Add to dashboard Cite

SUMMARY.We have developed a novel analytical method for serum lipoproteins using a commercially available capillary electrophoresis apparatus, BioFocus 3000 (Bio-Rad Laboratories Co Ltd, USA). The analytical principle is isotachophoresis (ITP), using a carrier ampholyte, BioLyte 7/9, as a spacer ion. The method allows a much higher resolution of lipoproteins than of amino acid mixtures. Serum lipoproteins are normally separated into 13±15 peaks, including some shoulder peaks. The reproducibility of repeated analysis within a day was relatively good with the coef®cient of variation within the range 0´9±1´1%. VLDL, LDL and HDL prepared by discontinuous density ultracentrifugation could be further separated by capillary ITP. This high-resolving ability of our method enabled detection of small amounts of abnormal lipoprotein species. For example, small dense LDL, which is thought to be an atherogenic lipoprotein, could be detected within the LDL group peak. Moreover, an abnormal HDL, apolipoprotein E-rich HDL, was also detected by a single analysis. These ®ndings suggest that our capillary ITP method is a useful means for detailed analysis of lipoproteins and thus for clinical diagnosis of hyperlipoproteinaemic subjects.

show abstract

Electrofocusing followed by gradient electrophoresis: A two-dimensional polyacrylamide gel technique for the separation of proteins and its application to the immunoglobulins

Cited by 29 publications

References 16 publications

The use of proteomics for the assessment of clinical samples in research

The use of proteomics for the assessment of clinical samples in research

Analysis of lipid hydrolytic activity by esterase on blotting membrane followed by separation using non‐denaturing two‐dimensional gel electrophoresis

Capillary isotachophoretic analysis of serum lipoproteins using a carrier ampholyte as spacer ion

Contact Info

Product

Resources

About