Downward Blotting of Proteins in a Model Based on Apolipoprotein(a) Phenotyping

B. Nagy, R. Costello, G. Csako

Research output: Article

5 Citations (Scopus)

Abstract

Standard immunoblotting ("Western blot") involves electrotransfer of proteins from a separation gel (usually acrylamide) onto a membrane. Recently, a downward capillary method with increased hybridization efficiency was developed for DNA and RNA. The present work assessed the applicability of this method to proteins in a model based on human apolipoprotein(a) [apo(a)] isoforms which consist of a single, >200-kDa polypeptide chain varying in size with a repeat sequence. After reduction treatment and sodium dodecyl sulfate-agarose gel electrophoresis, serum proteins were transferred from the gel by upward or downward (Turboblotter) capillary action onto nitrocellulose membranes in Tris-buffered saline, pH 7.5, at room temperature. Increased detectability of apo(a) isoforms was achieved by substituting comparatively high molar concentrations of protein A for true second antibody. With downward capillary transfer and short 37°C incubations, the apo(a) phenotyping could be completed in about 26 h and required less than 8 h effective processing time. The downward transfer was about twice as fast (complete within 1 h) as the upward version and with this speed it offers a good alternative to electroblotting as well.

Original languageEnglish
Pages (from-to)40-45
Number of pages6
JournalAnalytical Biochemistry
Volume231
Issue number1
DOIs
Publication statusPublished - okt. 1995

Fingerprint

Apoprotein(a)
Gels
Protein Isoforms
Capillary Action
Membranes
Proteins
Collodion
Agar Gel Electrophoresis
Acrylamide
Staphylococcal Protein A
Electrophoresis
Immunoblotting
Sodium Dodecyl Sulfate
Sepharose
Blood Proteins
Western Blotting
RNA
Peptides
Temperature
Antibodies

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology
  • Biophysics
  • Biochemistry

Cite this

Downward Blotting of Proteins in a Model Based on Apolipoprotein(a) Phenotyping. / Nagy, B.; Costello, R.; Csako, G.

In: Analytical Biochemistry, Vol. 231, No. 1, 10.1995, p. 40-45.

Research output: Article

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