Advertisement

Peroxisomes pp 25-36 | Cite as

Immunoblotting of Peroxisomal Proteins with Monospecific Antibodies

  • C. Pacot
Part of the Springer Laboratory book series (SLM)

Abstract

The most commonly used protein electrophoresis system today is the method described by Laemmli [2]. The Laemmli procedure, named sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), is a discontinuous system: the pretreated proteins are first concentrated in a stacking gel before entering the separating gel. Before electrophoresis, proteins are denaturated using SDS, an anionic detergent, that wraps around the polypeptide backbone and confers a negative charge to the polypeptide in proportion to its length. Proteins are also treated with a reducing agent such as 2-mercaptoethanol, which breaks disulfide bonds. After treatment, polypeptides are separated on the support gel, a Polyacrylamide matrix, on the basis of molecular weight by means of an electrical field in the presence of SDS. The major use of this system is to determine the molecular weight of polypeptides by running the gel silmutaneously with standard polypeptides of known molecular weights. A linear relationship exists between the log10 of the molecular weight of a polypeptide and its Rf (the distance from the limit between the stacking and separating gels to the polypeptide band, divided by the distance from the limit of the two gels to the front underlined by a low molecular weight dye).

Keywords

Plastic Dish Urate Oxidase Peroxisomal Protein Peroxisome Fraction Break Disulfide Bond 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Harlow E, Lane D (1988) In “Antibodies, A laboratory manual.”, Cold Spring Harbor Laboratory, New York, USAGoogle Scholar
  2. 2.
    Laemmli, EK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  3. 3.
    Nemali MR, Usuda N, Reddy MK, Oyasu K, Hashimoto T et al. (1988) Comparison of constitutive and inducible levels expression of peroxisomal β-oxidation and catalase genes in liver and extrahepatic tissues of rat. Cancer Res 48:5316–5324PubMedGoogle Scholar
  4. 4.
    Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from Polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Sci USA 76:4350–4354CrossRefGoogle Scholar
  5. 5.
    Van den Bosch H, Schutgens RBH, Wanders RJA, Tager JM (1992) Biochemistry of peroxisomes. Annu Rev Biochem 61:157–197PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1994

Authors and Affiliations

  • C. Pacot

There are no affiliations available

Personalised recommendations