Protein Analysis And Purification

by Rosenberg, Ian M.

• ISBN13: 9780817643416
• ISBN10: 0817643419
• Edition: 2nd
• Format: Paperback
• Copyright: 2004-12-01
• Publisher: Birkhauser

Summary

This comprehensive, practical, and user-friendly manual is designed to be a practical progression of experimental protocols that an inexperienced investigator may follow when embarking on a biochemical or biotechnology project. It will also appeal to experienced researches and graduate students as a benchtop handbook which, without sacrificing scientific concepts, maintains the spirit of experimentation.

Table of Contents

Preface	vii
Acknowledgments	ix
An Overview of This Manual	1	(7)
Introduction	1	(2)
Kits, Cores, and Computers	3	(1)
How to Use This Book	3	(1)
Basic Laboratory Equipment	4	(1)
Laboratory Automation	5	(1)
Beyond Protein Analysis and Purification	6	(2)
Protein Structure	8	(16)
Introduction	8	(1)
The Amino Acids	8	(3)
The Four Levels of Protein Structure	11	(6)
Primary Structure	11	(2)
Secondary Structure	13	(3)
Tertiary Structure	16	(1)
Quaternary Structure	17	(1)
Chemical Characteristics of Proteins	17	(7)
Hydrophobicity	19	(1)
Consensus Sequences	19	(2)
Proteomics	21	(3)
Tracking the Target Protein	24	(39)
Introduction	24	(2)
Labeling Cells and Proteins	26	(11)
Metabolic Labeling Cells in Culture	27	(1)
Metabolic Labeling Adherent Cells	28	(1)
Metabolic Labeling Cells Growing in Suspension	28	(1)
Pulse-Chase Labeling	29	(1)
Labeling Proteins Present at the Plasma Membrane	30	(1)
Lactoperoxidase Labeling Cell Surface Proteins	31	(1)
Labeling Surface Proteins with IODO-GEN®	32	(1)
Non-radioactive Biotinylation of Cell Surface Proteins	33	(1)
Domain-Selective Biotinylation and Streptavidin-Agarose Precipitation	34	(2)
Labeling Isolated Proteins with Chloramine T	36	(1)
Lysis---Preparation of the Cell Free Extract	37	(2)
Lysis Buffers	37	(1)
Lysis of Cells in Suspension (Continuation of Protocol 3.2)	38	(1)
Lysis of Adherent Cells (Continuation of Protocol 3.1)	39	(1)
Principles of Immunoprecipitation	39	(8)
Antibodies as Detection Tools	39	(1)
Polyclonal Antibodies	40	(1)
Monoclonal Antibodies	40	(1)
Antibody Based Analytical Techniques: Western Blotting and Immunoprecipitation	40	(2)
Immunoprecipitation	42	(2)
Protein Interaction Analysis	44	(1)
Sequential Immunoprecipitation: Dissociation and Reimmunoprecipitation of Immune Complexes	44	(2)
Eliminating Interfering Immunoglobulin Bands During IP-Western Detection: Analysis of the Immunoprecipitate under Non-Reducing Conditions	46	(1)
Nondenaturing Immunoprecipitation	47	(1)
Additional Methods to Identify Associated Proteins	47	(16)
Sucrose Gradients	48	(1)
Preparation of Sucrose Gradients	48	(3)
Fractionating a Sucrose Gradient	51	(2)
Chemical Cross-Linking	53	(1)
General Considerations for Cross-Linking	54	(1)
Cross-Linking Proteins Added to Cells: Analysis of Receptor--Ligand Interaction	55	(1)
Cross-Linking Proteins in Solution	55	(1)
Cross-Linking Extraneously Added Ligand to Cells	56	(1)
Cross-Linking Proteins in Solution Using the Homobifunctional Reagent Dithiobis (succinimidyl propionate) (DSP)	57	(1)
Analysis of Protein-Protein Interactions	58	(1)
Mapping Protein-Protein Contact Sites	58	(1)
Yeast Two-Hybrid Systems	59	(1)
Analyzing Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)	60	(3)
Electrophoretic Techniques	63	(55)
Introduction to Polyacrylamide Gel Electrophoresis (PAGE)	63	(3)
Preparation of SDS-Polyacrylamide Gels	66	(13)
Assembling the Plates	66	(1)
Choosing the Acrylamide Concentration	67	(1)
Casting the Separating Gel	67	(2)
Casting the Stacking Gel	69	(1)
Gradient Gels	70	(2)
Sample Preparation	72	(1)
Running the Gel: Attaching the Gel Cassette to the Apparatus and Loading the Samples	73	(3)
Drying the Gel	76	(1)
Separation of Low Molecular Weight Proteins by Tricine-SDS-PAGE (TSDS-PAGE)	77	(2)
Safety Considerations	79	(1)
2-Dimensional (2-D) Gel Systems	79	(16)
Isoelectric Focusing	81	(2)
Preparation of the Sample for Isoelectric Focusing	83	(1)
Single-Step Extraction/Solubilization Buffer	84	(1)
Preparation and Running of Isoelectric Focusing Tube Gels	84	(2)
Equilibration of the First-Dimension Gel or Strip	86	(1)
Flaws with 2-D Analysis	86	(1)
Measuring the pH of the Gel Slices	87	(2)
Nonequilibrium pH Gradient Electrophoresis (NEPHGE)	89	(1)
2-D Gels---The Second Dimension: SDS-PAGE	90	(1)
Fluorescence Two-Dimensional Difference Gel Electrophoresis (2-D DIGE)	91	(1)
Labeling Proteins with Cyanine Dyes (Cy3 and Cy5)	91	(1)
2-D PAGE Databases	92	(1)
Nonreducing-Reducing 2-D Gels	92	(3)
Detection of Protein Bands in Polyacrylamide Gels	95	(10)
Staining and Destaining the Gel with Coomassie Blue	96	(2)
Coomassie Staining Using GelCode® Blue	98	(1)
Staining Gels with SYPRO® Ruby	98	(1)
Viewing and Imaging a SYPRO Ruby-Stained 1-D or 2-D Gel	99	(1)
Silver Staining	100	(1)
Reversible Negative Staining of Proteins in Gels with Imidazole and Zinc Salts	101	(2)
Molecular Weight Determination by SDS-PAGE	103	(2)
Recovery of Proteins from the Gel	105	(1)
Excising the Protein Band from the Dried Gel	105	(1)
Extracting the Target Protein from the Dried Gel	106	(1)
Identification of Enzyme Activity in Polyacrylamide Gels	106	(12)
General Considerations	107	(1)
Localization of Proteases: Copolymerization of Substrate in the Separating Gel	107	(1)
Identification of Protease Inhibitors: Reverse Zymography	108	(1)
Locating the Enzyme Activity: Reacting the Gel with Substrate Solution after Electrophoresis	109	(1)
Detection of β-glucuronidase Activity in Polyacrylamide Gels	110	(1)
Identification of DNA Binding Proteins---Gel Shift Assay	111	(1)
Gel Shifts	112	(6)
Getting Started with Protein Purification	118	(35)
Introduction	118	(2)
Making a Cell Free Extract	120	(8)
Cellular Disruption	121	(1)
Extraction Buffer Composition	122	(1)
Protease Inhibitors	122	(1)
Methods of Cell Disruption	123	(2)
Clarification of the Extract	125	(1)
Nuclear Extracts	126	(1)
Total Lymphocyte Extract	127	(1)
Subcellular Fractionation	127	(1)
Subcellular Markers	128	(1)
Protein Quantitation	128	(8)
The Bradford Method	130	(1)
Bradford Standard Assay	130	(2)
Bradford Microassay	132	(1)
Protein Determination Using Bicinchoninic Acid (BCA)	133	(1)
Compatible Substances for the BCA Protein Assay	134	(1)
Incompatible Substances	134	(1)
NanoOrange® Protein Quantitation Assay: A Fluorescence-Based Assay of Proteins in Solution	135	(1)
Manipulating Proteins in Solution	136	(7)
Stabilization and Storage of Proteins	136	(1)
Concentrating Proteins from Dilute Solutions	137	(1)
Recovery of Protein by Ammonium Sulfate Precipitation	138	(1)
Ultrafiltration	138	(2)
Lyophilization	140	(1)
Dialysis	140	(1)
Preparation of Dialysis Tubing	141	(1)
Changing the Buffer by Gel Filtration	142	(1)
Precipitation Techniques	143	(10)
Salting Out with Ammonium Sulfate	143	(2)
Precipitation with Acetone	145	(1)
Precipitation with Polyethylene Glycol (PEG)	146	(1)
PEG Precipitation	146	(1)
Removal of PEG from Precipitated Proteins	147	(1)
Precipitation by Selective Denaturation	148	(1)
Recovery of Protein from Dilute Solutions by Methanol Chloroform Precipitation	148	(1)
Recovery of Protein by Trichloroacetic Acid (TCA) Precipitation	149	(1)
Concentration of Proteins by Acetone Precipitation	150	(1)
What to Do When All Activity Is Lost	150	(3)
Membrane Proteins	153	(24)
Introduction	153	(1)
Peripheral Membrane Proteins	154	(4)
Alkali Extraction	156	(1)
High pH Membrane Fractionation	157	(1)
Integral Membrane Proteins	158	(2)
Organic Alcohol Extraction of Peripheral Membrane Proteins	158	(1)
Butanol Extraction	159	(1)
Single-Phase Butanol Extraction	159	(1)
Detergents	160	(17)
Properties of Detergents	165	(1)
Critical Micelle Concentration (CMC)	165	(1)
Micelle Molecular Weight	165	(1)
Hydrophile-Lipophile Balance (HLB)	166	(1)
Classification of Detergents	166	(1)
Ionic Detergents	166	(1)
Nonionic Detergents	166	(1)
Bile Salts	166	(1)
Detergent Solubilization	167	(1)
Choosing a Detergent	168	(1)
Choice of Initial Conditions	169	(1)
Differential Detergent Solubilization	169	(2)
Solubilization Trial	171	(1)
Protein-to-Detergent Ratio	172	(1)
Detergent Removal	173	(1)
Removal of Ionic Detergents	173	(1)
Removal of Nonionic Detergents	173	(1)
Extracti-Gel® D	173	(4)
Transfer and Detection of Proteins on Membrane Supports	177	(34)
Introduction	177	(1)
Transfer of Proteins to Membrane Supports	177	(6)
Transfer of Proteins to Nitrocellulose or Polyvinylidene Difluoride	178	(3)
Troubleshooting Western Blots	181	(1)
Enhanced Capture of Small Histidine Containing Polypeptides on Membranes in the Presence of ZnCl2	181	(1)
Dot Blots	182	(1)
Thin-Layer Chromatography Blotting	182	(1)
Staining the Blot	183	(3)
Total Protein Staining with India Ink	183	(1)
Reversible Staining with Ponceau S	184	(1)
Irreversible, Rapid Staining with Coomassie Brilliant Blue	184	(1)
Staining Immobilized Glycoproteins by Periodic Acid/Schiff (PAS)	185	(1)
Recovery of Proteins from the Blot	186	(10)
Recovery of Proteins Using an Organic Solvent System	186	(1)
Recovery of Proteins from the Blot Using a Detergent-Based Solvent System	187	(1)
Blocking the Blot	188	(1)
Exposing the Blot to Primary Antibody	188	(2)
Ligand Blotting	190	(1)
Southwestern Blotting	191	(1)
Far Western Blotting	191	(1)
Ligand Binding	192	(1)
Lectin Blotting	193	(2)
Bacterial Protein Overlay Analysis	195	(1)
Detection of the Target Protein	196	(15)
Protein A	196	(1)
Second Antibody Conjugate	197	(1)
Biotin Avidin System	197	(1)
Biotinylation of Proteins	197	(1)
Purifying and Biotinylating Antibodies from Immunoblots	198	(1)
Enzymatic Detection Methods	199	(1)
Horseradish Peroxidase	199	(1)
Colorimetric Detection with Diaminobenzidine, 3,3',4,4'-tetraaminobiphenyl) (DAB)	199	(1)
Colorimetric Detection Using Alkaline Phosphatase	200	(1)
Enhanced Chemiluminescence	201	(2)
Stripping and Reprobing the Blot	203	(1)
Detection of Radiolabeled Proteins	203	(1)
Direct Autoradiography	204	(1)
Removing Unwanted Background Signal from X-ray Film	205	(2)
Phosphorimaging	207	(4)
Identification of the Target Protein	211	(48)
Introduction	211	(1)
Peptide Mapping	211	(3)
Thermal Denaturation	212	(1)
Preparing the Target Protein for Digestion	213	(1)
Enzymatic Cleavage of Proteins	214	(5)
Peptide Mapping by Proteolysis and Analysis by Electrophoresis	215	(2)
Cleavage of Proteins Transferred to PVDF or NC Membranes	217	(1)
Cleavage of Proteins Immobilized on the Membrane	217	(1)
Tryptic Cleavage of Protein Eluted from PVDF Membrane	218	(1)
Chemical Cleavage	219	(6)
Cyanogen Bromide Cleavage of Proteins on PVDF Membrane	219	(2)
N-Chlorosuccinimide (NCS) Mapping	221	(1)
Hydroxylamine Cleavage of Proteins in Polyacrylamide Gels	222	(1)
Formic Acid Cleavage	223	(1)
Chemical Cleavage at Cysteine Residues with DTNB	224	(1)
Microsequencing from PVDF Membranes	225	(34)
When, and In What Form Do You Submit the Target Protein to the Protein Sequencing Core?	227	(1)
Transferring Spots from 2-D Gels to PVDF Membranes	228	(1)
Sequencing Glycopeptides	228	(1)
Protein Hydrolysis: Total Amino Acid Composition of the Target Protein	229	(1)
Identifying Proteins Using Mass Spectrometry	230	(1)
Preparation of Proteins for MS Analysis	231	(1)
Partial Proteolysis	231	(1)
In-gel Tryptic Digestion	232	(1)
Extraction of Peptides from Gel Pieces Containing Integral Membrane Proteins	233	(1)
Elution of Target Protein from SDS-PAGE	233	(1)
Protein Transfer to a Membrane	234	(1)
Considerations	234	(1)
MS Basics	234	(1)
Electrospray and Tandem Mass Spectrometry	235	(2)
MALDI and Peptide-Mass Mapping	237	(1)
Post-Source Decay (PSD) MALDI-MS	237	(1)
Protein Identification by MS	238	(1)
Peptide Mass Fingerprint Analysis	238	(1)
Peptide Fragmentation	239	(1)
Peptide Ladder Sequencing	240	(1)
Peptide Sequence Tag	240	(1)
Identification of a Gene Product	240	(1)
Posttranslational Modifications and MS	241	(2)
Caveats When Using MS	243	(1)
Protein Database Searches	244	(1)
Bioinformatics	244	(1)
The Rise of Biological Databases	245	(1)
Search Engines	245	(1)
Databases	246	(2)
Identifying a Target Protein	248	(1)
Gene Analysis Tools	248	(1)
Subcellular Localization	249	(1)
Protein Domain Families	250	(1)
Structural Classification of Proteins	251	(1)
Genomic Organization	252	(1)
Additional Useful Sites on the Internet	252	(1)
PCR Primer Design Programs	252	(1)
Microarrays and Data Mining---the Challenge of Data Analysis	253	(6)
Identifying and Analyzing Posttranslational Modifications	259	(65)
Introduction	259	(2)
Glycosylation	261	(17)
Chemical Deglycosylation Using Trifluoromethanesulfonic Acid (TFMS)	265	(2)
N-Glycosylation	267	(1)
Removal of the Oligosaccharide from the Glycoprotein with N-Glycanase	267	(1)
N-glycosidase F (GPase F) Treatment of Glycoproteins in Immunoprecipitates	268	(1)
Tunicamycin	269	(1)
O-Glycosylation	270	(1)
Identification of O-Glycosylated Amino Acids by Alkaline β-Elimination	270	(1)
β-Elimination of O-Glycans from Glycoproteins Immobilized on Blots	271	(1)
O-Glycosidase	271	(1)
O-Glycanase	272	(1)
Combined Use of N-Glycanase and O-Glycanase	272	(1)
Endoglycosidase H	272	(1)
Neuraminidase (NA)	273	(1)
Desialylation with Clostridium perfringens Neuraminidase	274	(1)
Desialylation with Arthrobacter ureafaciens Neuraminidase	275	(1)
Lectins as Tools for Carbohydrate Analysis	276	(1)
Proteoglycans	277	(1)
Is the Target Protein a Proteoglycan?	277	(1)
Phosphorylation	278	(24)
Metabolic Labeling of Cells with [32P]orthophosphate	280	(1)
Can the Target Protein Be Phosphorylated?	281	(1)
Determination of the Type of Phosphorylated Amino Acid-Immunoblotting with Anti-Phosphoamino Acid Antibodies	282	(1)
Phosphorylation of Membrane Proteins with [γ-32P]GTP	283	(1)
Enzymatic Dephosphorylation	283	(1)
Potato Acid Phosphatase	283	(2)
Alkaline Phosphatase	285	(1)
Immune Complex Kinase	285	(2)
Renaturation of Immobilized Kinases on PVDF Membranes	287	(2)
Phosphorylation of Substrates in SDS-Gels	289	(1)
Phosphopepetide and Phosphoamino Acid Analysis	290	(1)
One-Dimensional Phosphopeptide Mapping	291	(1)
Two-Dimensional Phosphopeptide Mapping	291	(1)
Isolation of Phospho-Proteins from SDS Gels: Preparation for Phosphopeptide Mapping	292	(1)
Tryptic Digestion of Isolated Phosphoproteins	293	(1)
Applying the Sample to the TLC Plate and Electrophoresis in the First Dimension	294	(1)
Second Dimension: Thin-Layer Chromatography	295	(1)
Isolation of Individual Phosphopeptides from TLC Plates	296	(1)
Phosphoamino Acid Analysis	296	(2)
Phosphoamino Acid Analysis of Phosphoproteins Isolated from PVDF Membranes	298	(1)
Identification of Phosphohistidine Residues Following Heat Treatment	299	(1)
Treatment with Diethyl Pyrocarbonate	300	(1)
Treatment of Phosphorylated Membranes with HCI and NaOH	300	(1)
Sulfation	301	(1)
Lipid Modification of Proteins	302	(12)
Palmitoylation and N-Myristoylation of Proteins	303	(1)
Analysis of Bound Fatty Acids	304	(1)
Identification of Palmitoylated and Myristoylated Proteins	304	(1)
Isoprenylation	305	(1)
Metabolic Labeling with [3H]Mevalonic Acid Derivatives	306	(2)
Enzymatic Prenylation of Recombinant Proteins	308	(1)
Glypiation	308	(2)
Is the Target Protein Glycosyl Phosphatidylinositol Anchored?	310	(1)
Use of Triton X-114	310	(1)
Preparation of Triton X-114	310	(1)
Fractionation of Integral Membrane Proteins with Triton X-114	311	(1)
Digestion with Phosphatidylinositol Specific Phospholipase C (PI-PLC)	312	(1)
Metabolic Labeling with Precursors of the GPI Structure	313	(1)
Use of Anti-CRD	313	(1)
Selected Modifications	314	(10)
Transamidation	314	(1)
Acetylation	314	(1)
Methylation	314	(1)
Hydroxylation of Proline and Lysine	315	(1)
Degradation	315	(1)
Ubiquitination	315	(1)
Proteolytic Processing	316	(8)
Chromatography	324	(61)
Introduction	324	(1)
Important Terminology Used in Chromatography	325	(1)
Gel Filtration Chromatography	326	(12)
Choice of Buffer	329	(1)
Choice of Column Size	329	(1)
Preparation of the Gel: Hydrating and Degassing	330	(1)
Packing the Column	331	(1)
Flow Rate	332	(1)
Hydrostatic Pressure	333	(1)
Sample Application	334	(1)
Loading Sample onto a Drained Bed	334	(1)
Loading Sample Under the Eluent	335	(1)
Making Sure the Column Does Not Run Dry	335	(1)
Molecular Weight Determination	335	(1)
Spin Columns Used in Gel Filtration	336	(1)
Spin Columns	337	(1)
Testing Fractions to Locate Protein: Bradford Spot Test	338	(1)
Introduction to HPLC	338	(4)
Packing Materials	339	(1)
Column Designs	340	(1)
Column Guards	340	(1)
Detectors	340	(1)
Choosing the Right Conditions---Some Helpful Tips	341	(1)
HPLC---Size Exclusion	342	(1)
Ion Exchange Chromatography: Separation on the Basis of Charge	342	(14)
Simplified Theory of Ion Exchange	343	(1)
Functional Groups on Exchange Columns	344	(1)
Choice of Exchanger Matrix	345	(1)
Preparation of the Exchanger	346	(1)
Choice of Buffer	346	(1)
Batch Adsorption	347	(1)
Selecting the Starting pH	348	(1)
Packing an Ion Exchange Column	348	(1)
Experimental Tips	349	(1)
Elution-Step or Linear Gradient?	349	(2)
Regeneration of Sephadex Ion Exchangers	351	(1)
Regeneration of Sepharose Ion Exchangers	351	(1)
Chromatofocusing	351	(2)
Removing the Polybuffer	353	(1)
HPLC-Ion Exchange Chromatography	353	(1)
Membrane Adsorbers	354	(1)
Perfusion Chromatography	355	(1)
Hydrophobic Interaction Chromatography (HIC)	356	(7)
Simplified Theory of HIC	356	(1)
Protein Fractionation by HIC	357	(1)
Solid Phase Extraction Cartridges	358	(1)
Reversed Phase HPLC	359	(2)
Reversed Phase HPLC for the Isolation of Peptides	361	(1)
Multidimensional Liquid Chromatography	362	(1)
Affinity Chromatography	363	(22)
Immunoaffinity Purification	364	(1)
Direct Antibody Coupling to Protein A Beads	365	(1)
Indirect Antibody Coupling to Protein A Beads	366	(1)
Preparation of Affinity Columns	367	(1)
Flow Rate	368	(1)
Binding Antigens to Immunoaffinity Matrices	369	(1)
Nonspecific Interactions	369	(1)
Blocking the Affinity Matrix	370	(1)
Elution of Antigens from Immunoaffinity Matrices	370	(2)
Eluting the Antigen	372	(1)
Ligand Affinity Chromatography	373	(1)
Immobilization of Proteins to N-Hydroxysuccinimide Ester Derivatives of Agarose	373	(2)
Toluene Sulfonyl Chloride (Tosyl Chloride)	375	(1)
Pseudo-Affinity Adsorbents	375	(2)
Lectin Affinity Chromatography	377	(1)
Glycoprotein Purification Using Wheat Germ Agglutinin (WGA)	378	(1)
Immobilized Metal Chelate Chromatography (IMAC)	379	(1)
Purifying a Histidine Tagged Recombinant Fusion Protein	380	(1)
Hydroxylapatite Chromatography	381	(4)
Recombinant Protein Techniques	385	(46)
Introduction	385	(1)
Recombinant Protein for Antibody Production	386	(1)
Protein for Biochemical or Cell Biological Studies	387	(1)
In vitro Transcription and Translation	388	(8)
Preparation of the DNA Template	388	(1)
In vitro Transcription---Preparation of the mRNA	389	(1)
Guanylyltransferase Catalyzed Addition of a G(5')ppp(5')G Cap to mRNA	390	(1)
In vitro Translation: Protein Synthesis	391	(1)
Cotranslational Processing Using Canine Pancreatic Microsomal Membranes	392	(1)
Translocated Products Are Resistant to Protease Digestion	393	(1)
Was the Translational Product Glycosylated? Endoglycosidase H (Endo H) Analysis	394	(1)
Protein Transduction: A Method for Introducing Exogenous Proteins into Cells	395	(1)
Recombinant Gene Products in E. coli: Expression, Identification and Characterization	396	(6)
Expression and Purification of lacZ and trpE Fusion Proteins	397	(1)
lacZ Induction	398	(1)
Induction of the trpE Fusion Protein	398	(1)
Preparation of the Protein Extract	399	(1)
Solubilization of the Fusion Protein	400	(1)
Purification of Eukaryotic Proteins from Inclusion Bodies in E. coli	400	(2)
Affinity Tags	402	(13)
Removing the Tag	403	(1)
Glutathione-S-Transferase (GST) Fusion Proteins	404	(1)
Production and Analysis of GST Fusion Protein Transformants (Small Scale)	405	(1)
Purification of GST Fusion Proteins	406	(1)
Removing the GST from the Fusion Protein	407	(1)
His-Tag Purification System	408	(2)
Maltose Binding Protein (MBP) Fusion Proteins	410	(3)
Staphylococcal Protein A and ZZ	413	(2)
Green Fluorescent Protein (GFP)	415	(1)
Expression of Foreign Proteins in Eukaryotic Cells	415	(16)
Expression and Isolation of Recombinant Proteins from Yeast	415	(1)
Preparation of Protein Extracts from Yeast	416	(2)
Expression of Proteins in Insect Cells Using Baculoviral Vectors	418	(1)
Expression of Foreign Proteins in Mammalian Cells	419	(1)
Transfection: Expression of Recombinant Proteins in Eukaryotic Systems	420	(1)
Transfection of DNA into Eukaryotic Cells with Calcium Phosphate	421	(1)
Glycerol Shock	422	(1)
Transfection Using DEAE-Dextran	423	(1)
Stable Transfections	424	(1)
Picking Stable Colonies	425	(6)
Appendices	431	(80)
A. Safety Considerations	433	(2)
First Aid: Emergency Procedures	433	(2)
B. Antibody Preparation	435	(6)
Production of Polyclonal Antisera in Rabbits	435	(1)
Preparation of the Antigen-Adjuvant Emulsion	436	(1)
Intramuscular Immunization (IM)	436	(1)
Intradermal Immunization	437	(1)
Subcutaneous Immunization	437	(1)
Bleeding the Rabbit and Serum Preparation	437	(1)
Precipitation of IgG with Saturated Ammonium Sulfate	438	(1)
Purification of Antibody Using Protein A Affinity Columns	439	(1)
Purifying Total Ig	439	(1)
Numbering Mice	440	(1)
C. Solutions	441	(10)
Commercial Strengths of Common Laboratory Chemicals	441	(1)
Water	441	(1)
Molarity	442	(1)
Choosing and Preparing Buffers	442	(5)
Common Laboratory Solutions	447	(2)
Extinction Coefficients	449	(2)
D. Nucleic Acids	451	(5)
Spectrophotometric Conversions	451	(1)
DNA/Protein Conversions	451	(1)
Oligonucleotide Concentrations	451	(1)
Fluorometric Estimation of DNA Concentrations	451	(1)
RNA Precipitation	452	(4)
E. Modifications and Motifs	456	(9)
Nomenclature	456	(2)
Protein Modification Sequences	458	(2)
Protein Kinase Recognition Sequence Motifs	460	(1)
Subcellular Localization Motifs	461	(1)
Protein Databases	462	(3)
F. Centrifugation	465	(15)
Nomogram	467	(2)
General Purpose Centrifuge Rotors	469	(4)
Ultracentrifuge Rotors	473	(7)
G. Proteases and Proteolytic Enzyme Inhibitors	480	(10)
Commonly Used Proteases	480	(2)
Preparation of Defatted BSA	482	(1)
Protease Inhibitors	483	(7)
H. Radioactivity	490	(3)
Manual and Machine Film Processing	491	(2)
I. Tissue Culture	493	(2)
Transwell Permeable Supports	493	(2)
J. Miscellaneous	495	(7)
Siliconizing Glassware	495	(1)
Unit Prefixes	495	(1)
The Greek Alphabet	496	(1)
Abbreviations	496	(3)
HPLC Pump Pressure Conversion	499	(1)
Dipeptide Masses	500	(1)
Mass Differences Considered in Molecular Weight Analysis of Proteins	501	(1)
K. List of Suppliers, Vendors, Manufacturers	502	(9)
Index	511