Subcellular Fractionation A Practical Approach

by Graham, John; Rickwood, David

• ISBN13: 9780199634958
• ISBN10: 0199634955
• Format: Hardcover
• Copyright: 1997-03-27
• Publisher: Oxford University Press

Summary

Many investigations into the structure and function of cells and tissues require the isolation of a particular membrane or subcellular component (organelle). This book covers all the necessary aspects, from the homogenization of cells via a variety of separation techniques, to the isolation and characterization of peroxisomes and other organelles. Basic separation techniques needed in all cell biology laboratories are given and presented in the context of a number of related topics such as the labelling of ligands, the kinetics of their internalization and analysis of data, and the electron microscopy of organelles. Subcellular Fractionation contains expert advice and detailed methodology, and will be invaluable to researchers in this important area.

Table of Contents

List of contributors	xvii	(2)
Abbreviations	xix
1. Homogenization of tissues and cells	1	(30)
John M.Graham
1. Introduction	1	(1)
2. Aims of the homogenization procedure	1	(2)
3. Influence of sample type	3	(1)
4. Homogenization media	4	(2)
5. Methods of homogenization	6	(8)
Type 1 homogenizers	7	(3)
Type 2 homogenizers	10	(4)
6. Homogenization of tissues and cells	14	(14)
Mammalian liver	14	(3)
Brain	17	(1)
Muscle	18	(1)
Mammalian tissue culture cells	19	(3)
Plant organelles	22	(1)
Yeast	23	(1)
Other fungi and algae	24	(1)
Trypanosomes	25	(1)
Bacteria	26	(2)
References	28	(3)
2. Isolation of subcellular fractions	31	(40)
Richard H. Hinton
Barbara M. Mullock
1. Introduction	31	(3)
2. Cmposition of a tissue homogenate	34	(4)
3. Properties of cell organelles	38	(2)
Factors affecting organelle density and size	38	(2)
4. Centrifugal methods for the separation of organelles	40	(10)
Separation by size	41	(2)
Separation by density	43	(6)
Density perturbation	49	(1)
5. Non-centrifugal procedures	50	(1)
Immunsiolation	50	(1)
Separation by electrophoresis	50	(1)
6. Identification of separated material	51	(4)
Market enzymes	51	(3)
Introduced markers for endocytic and exocytic pathways	54	(1)
Characteristic non-enzymatic proteins	54	(1)
7. Assessment of the purity of fractions	55	(3)
Purity and purification	56	(1)
Problems from cell heterogeneity within tissues	56	(1)
Problems arising from organelle fragmentation	57	(1)
Missorting in the exocytic and endocytic pathway	57	(1)
8. Fractionation problems	58	(2)
No separation	58	(1)
Aggregation following resuspension of a fraction	58	(1)
Poor recovery of markers	58	(2)
Damage to cell structures	60	(1)
9. A systematic approach to cell fractionation	60	(9)
Preliminary studies	61	(1)
Determination of the properties of compoents of the homogenate	61	(2)
Method development	63	(2)
Simplification of the separations	65	(4)
References	69	(2)
3. Isolation and characterization of nuclei and nuclear subfraction	71	(31)
David Rickwood
Anthea Messent
Dipak Patel
1. Introduction	71	(1)
2. Methods of preparing purified nuclei	71	(7)
Types of cell and tisue samples	71	(1)
Homogenization media	72	(1)
Homogenization methods	73	(1)
Centrigfuation conditions	73	(4)
Assays of nuclear purity	77	(1)
3. Methods for purifying metaphase chromosomes	78	(2)
4. Isolation of nuclear subfractions	80	(6)
Preparation of nucleoli	80	(1)
Preparation of nuclear membranes	81	(3)
Isolation of nuclear matrix	84	(1)
Preparation of nucleoids	85	(1)
5. Isolation of nucleoprotein complexes	86	(3)
Isolation of polynucleosomes of chromatin	86	(2)
Ribonucleoproteins	88	(1)
6. Isolation of nuclear macromolecules	89	(10)
Isolation of nuclear proteins	89	(2)
Isolation of nuclear RNA	91	(6)
Isolation of DNA	97	(2)
7. Functional assays of nuclei	99	(8)
Analysis of DNA-binding proteins	99	(2)
Transcription assays	101	(2)
References	103	(4)
4. Sybcellular fractionation of mitochondria	107	(36)
J. E. Rice
J. G. Lindsay
1. Introduction	107	(1)
2. Purification of mitochondria from various eukaryotic sources	108	(11)
Introduction	108	(4)
Protocols for purification of mitochondria from several eukaryotic sources	112	(7)
Further purification of mitochondrial fractions	119	(1)
3. Determination of mitochondrial purity	119	(11)
Introduction	119	(1)
Use of the oxygen electrode to determine mitochondrial integrity	120	(7)
Determination of the integrity of the mitochodrial outer membrane	127	(1)
Glucose hexokinase trap method for estimation of P:O ratios	128	(2)
4. Subfractionation of mitochondria	130	(6)
Preparation of submitochondrial particles by sonication	130	(1)
Preparation of mitoplasts using digitonin	131	(2)
Separation of `right side out'and `inside out' by submitochondrial particles by affinity chromatography on CNBr-cytochrome c coupled Sepharose	133	(2)
Assaying of mitochondrial marker enzymes	135	(1)
5. Medical aspects of mitochondrial research	136	(5)
Mitochondrial biogenesis	136	(1)
Oxidative phosphorylation and the electron transport chain	137	(1)
Mitochondrial myopathies	138	(1)
Techiques applied in the investigation of mitochondrial abnormalities	139	(2)
6. Concluding remarks	141	(1)
Reference	142	(1)
5. Isolation and characterization of peroxisomes	143	(26)
A. Volkl
E. Baumgart
H. D. Fahimi
1. Introduction	143	(1)
2. Preparation and purification of rat hepatic PO	144	(9)
General considerations	144	(1)
Experimental design	145	(5)
Analysis of results	150	(3)
3. Subfractionation of purified rat hepatic peroxisomes	153	(2)
Extraction of soluble matrix proteins	153	(1)
Extraction of core-bound urate oxidase	153	(1)
Isolation of integral membrane proteins of PO	153	(2)
4. Comparative characterization of isolated hepatic peroxisomes from different species	155	(3)
Enzyme activities of PO fractions	155	(2)
Immunoblotting of PO fractions	157	(1)
5. Embedding of PO fractions for electron microscopy (EM)	158	(7)
Equipment and solutions	158	(3)
Processing of PO fractions for EM	161	(4)
Results	165	(1)
Acknowledgements	165	(1)
References	165	(4)
6. Lysosomes and endocytosis	169	(36)
Tor Gjoen
Trond Olav Berg
Trond Berg
1. Current modles of endocytosis	169	(1)
2. Analysis of endocytic uptake of ligands and pinocytosis markers	170	(7)
Receptor-mediated endocytosis	170	(6)
Fluid phase endocytosis	176	(1)
3. Endocytosis of plasma membrane components	177	(1)
4. Analysis of intracellular transport of ligands/markers and endocytic receptor by means of subcellular fractionation	178	(7)
Differential centrifugation	178	(2)
Isopycnic centrifugation in gradients of sucrose, Nycodenz, or Percoll	180	(5)
Continuous flow electrophoresis (CFE)	185	(1)
5. Labelling of ligands and markers	185	(4)
Use of [125I]	185	(1)
Use of residualizing labels	186	(2)
Labelling of receptors	188	(1)
6. Markers for organelles involved in endocytosis	189	(3)
The plasma membrane	189	(2)
Endosomes	191	(1)
The Iysosomes	191	(1)
7. Induction of density shift of components of the endocytic pathway to determine a possible co-localization of ligand and other markers	192	(4)
Density shift induced in endsomes that contain ligand in complex with horse-radish peroxidase or colloidal gold	193	(3)
8. Use of specific perturbants to accumulate ligands and/or receptors at defined intracellular sites	196	(2)
9. Use of Iysosomal enzyme substrates to disrupt lysosomes selectively	198	(2)
10. Concluding remarks	200	(1)
References	200	(5)
7. The membranes of the secretory and exocytic pathways	205	(38)
John M. Graham
1. Introduction	205	(3)
Gradient media	205	(3)
2. Isolation of plasma membrane, endoplasmic reticulum, and Golgi from rat liver	208	(17)
Plasma membrane	208	(3)
Separation of plasma membrane domains	211	(1)
Endoplasmic reticulum (ER)	212	(2)
Golgi membranes	214	(7)
Isolation of membranes by immunoaffinity	221	(3)
Density perturbation methods	224	(1)
3. The secretory pathway	225	(14)
Resoulution of Golgi domains	225	(4)
Identification of vesicles involved in transport to the Golgi	229	(2)
Identification of vesicles involved in transport from the Golgi	231	(2)
In vitro systems to study transfer of molecules between membranes	233	(1)
Sorting of proteins destined for specific plasma membrane domains	234	(2)
Secretory pathways in yeast	236	(2)
Gel filtration	238	(1)
References	239	(4)
8. Isolation and purification of functionally intact mitochondria and chloroplasts from plant cells	243	(28)
Anthony L. Moore
David G. Whitehouse
1. Introduction	243	(1)
2. Mitochondria	243	(6)
General isolation principles	245	(4)
3. Chloroplasts	249	(11)
General isolation principles	251	(9)
4. Assays	260	(8)
Mitochondria	260	(5)
Chloroplasts	260	(5)
Acknowledgements	268	(1)
References	268	(3)
9. Ribosomes and polysomes	271	(32)
Ulrich A. Bommer
Nils Burkhardt
Ralf Junemann
Christian M. T. Spahn
Fracisco J. Triana-Alonso
Knud H. Nierhaus
1. Introduction	271	(1)
2. Isolation of ribosomes and polysomes	271	(16)
Prokaryotic ribosomes	271	(9)
Eukaryotic ribosomes	280	(7)
Assay systems	287	(13)
Protein synthesis systems	287	(5)
Testing partial reactions of the ribosome cycle	292	(8)
Acknowledgements	300	(1)
References	300	(3)
10. Electron microscopy of organelles	303	(26)
Nigel James
1. Introduction	303	(1)
2. Fixation	304	(8)
Importance of fixation	304	(1)
Chemical fixation	305	(2)
Osmolarity of fixative solutions	307	(1)
Temperature, pH, and other factors	308	(2)
Cryofixation	310	(1)
Cryoultramicrotomy	311	(1)
Freeze-fracture preparations	312	(1)
3. Embedding	312	(2)
Hardeners, modifiers, and catalysts	314	(1)
4. Microtomy	314	(2)
5. Staining	316	(10)
The electron microscope family	316	(1)
Iamge contrast	316	(2)
Structural staining	318	(1)
Antibody staining	319	(1)
Colloidal gold	319	(2)
Enzyme cytochemistry	321	(2)
Histochemistry	323	(2)
Negative staining	325	(1)
6. Further reading	326	(1)
References	327	(2)
A1. Core list of usppliers	329	(4)
Index	333