Pelleting Technique for Subcellular Fraction Electron Microscopy and/or Immunoelectron Microscopy
Subcellular fraction identification of RER, SER, Golgi, mitochondria, peroxisomes, melanosomes, etc. can be examined ultrastructurally using a pellet technique. Purified fractions are pelleted at 150,000 x g for approximately 30 minutes and fixed as a suspension in 2.5% buffered glutaraldehyde for three hours. The pellet is rinsed in buffer, resuspended in a 1:1 (v/v) with 3.0% agarose. This mixture solidifies at room temperature and is processed like a tissue specimen for electron microscopic examination and photography. A variation of this technique is available for immunogold labeling of subcellular fractions.
Electron micrograph of smooth (top) and rough
(bottom) endoplasmic reticulum
Ref: Phung, T.H., Roncome, A., de Mesy Jensen (Bentley), K.L., Sparks, C.E. and Sparks, J. D.: Phosphoinositide 3-kinase activity is necessary for insulin-dependent inhibition of apolipoprotein B secretion by rat hepatocytes and localized to the endoplasmic reticulum. J. Biol. Chem. 272:30693, 1997.
Subcellular fraction of rat heart mitochondria
embedded in Lowicryl resin. Sectioned and
labelled for ryanodine receptor with 15nm gold tag.
(Dr. Gisella Beutner et al,
J Biol Chem. 2001. 276(24):21482-8.)
Karen de Mesy Bentley, M.S., Director
Phone: (585) 275-1954
EM Labs: B-7811, B-7819