Immunoblot affinity purification

D. L. Spector, R. D. Goldman, L. A. Leinwand, E. Harlow, D. Lane

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

The immunoblotting method has evolved from early stages when antibodies were used to ‘stain’ polyacrylamide gels directly1, 2 to more versatile methods using replica techniques, in which the separated polypeptides are transferred to nitrocellulose membranes, chemically activated paper or nylon sheets. Although there are several variations on this basic theme, the most common and effective is electrophoretic transfer to nitrocellulose sheets3, 4. The separated proteins can then be probed with antibodies; this variation of the technique is known as immunoblotting (or western blotting). The membrane can also be probed with specific ligands, such as DNA, protein, small molecules (for example, heparin or GTP) or even whole cells. Electrophoretic transfer can be achieved either in a tank3 or in a semidry apparatus5, in which the buffer volume is reduced to filter paper pads. The following protocol presents the original method used for tank blotting.

Original languageEnglish (US)
Number of pages1
JournalNature Methods
Volume2
Issue number10
DOIs
StatePublished - Jan 1 2005

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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