Techniques

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, 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.