TY - JOUR
T1 - The isolation of large quantities of undamaged cellular organelles and cytosolic enzymes using a low-shear continuous tissue homogenizer
AU - Spelsberg, Thomas C.
AU - Reinhart, Gregory D.
AU - Barham, Steve
N1 - Funding Information:
The authors thank Ms. Barb Gosse, Ms. Kay Rasmussen, Mr. Bill Burger& Ms. Betty Thorpe, Mr. David Mork, Mr. Al Wussow, and Mr. Gordon Smith for their excellent technical and engineering assistance. This work was supported in part by Grants HD 9140 B and GM 30884 and the Mayo Foundation.
PY - 1984/12
Y1 - 1984/12
N2 - There is often a need to isolate large quantities of subcellular components such as membrane-coated organelles (e.g., nuclei, lysosomes, and mitochondria), cell membranes, and soluble (cytosolic) proteins. Instruments which can homogenize relatively large masses of tissue, primarily those with rapidly rotating blades and cylinders, are excessively vigorous, often resulting in damaged and/or low yields of the subcellular components. This paper describes procedures for obtaining high yields of undamaged subcellular components using a continuous bulk tissue homogenizer which performs with low shear (the low-shear continuous homogenizer or LSC). This homogenizer is simple in operation, durable and can be used with a variety of tissues. Fibrous tissues are more difficult to homogenize using this instrumentation and require a premincing to small pieces (0.2 to 1.0-cm diam) followed by filtration through 2-4 mesh (two to four apertures per inch). Methods for bulk preparations with enhanced recoveries of undamaged nuclei, and a typical soluble multimeric enzyme, phosphofructokinase, are presented. Electron microscope views of the homogenates show the preserved state of the other subcellular components. The LSC homogenizer requires less physical effort with no "hands on" operation and thus is safer. This homogenizer requires less homogenization time compared to the smaller, hand-held Potter-Elvehjem-type homogenizers. Operations requiring low temperature can be performed at room temperature as long as the continuously passing homogenate solutions are kept chilled.
AB - There is often a need to isolate large quantities of subcellular components such as membrane-coated organelles (e.g., nuclei, lysosomes, and mitochondria), cell membranes, and soluble (cytosolic) proteins. Instruments which can homogenize relatively large masses of tissue, primarily those with rapidly rotating blades and cylinders, are excessively vigorous, often resulting in damaged and/or low yields of the subcellular components. This paper describes procedures for obtaining high yields of undamaged subcellular components using a continuous bulk tissue homogenizer which performs with low shear (the low-shear continuous homogenizer or LSC). This homogenizer is simple in operation, durable and can be used with a variety of tissues. Fibrous tissues are more difficult to homogenize using this instrumentation and require a premincing to small pieces (0.2 to 1.0-cm diam) followed by filtration through 2-4 mesh (two to four apertures per inch). Methods for bulk preparations with enhanced recoveries of undamaged nuclei, and a typical soluble multimeric enzyme, phosphofructokinase, are presented. Electron microscope views of the homogenates show the preserved state of the other subcellular components. The LSC homogenizer requires less physical effort with no "hands on" operation and thus is safer. This homogenizer requires less homogenization time compared to the smaller, hand-held Potter-Elvehjem-type homogenizers. Operations requiring low temperature can be performed at room temperature as long as the continuously passing homogenate solutions are kept chilled.
KW - bulk isolation
KW - continuous homogenizer
KW - electron microscopy
KW - enzymes
KW - nuclei
KW - organelles
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U2 - 10.1016/0003-2697(84)90659-6
DO - 10.1016/0003-2697(84)90659-6
M3 - Article
C2 - 6241816
AN - SCOPUS:0021681224
SN - 0003-2697
VL - 143
SP - 237
EP - 248
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -