Protein analysis and purification: benchtop techniques
Saved in:
| Main Author: | |
|---|---|
| Format: | Book |
| Language: | English |
| Published: |
Boston [u.a.]
Birkhäuser
2005
|
| Edition: | 2. ed. |
| Subjects: | |
| Online Access: | Inhaltsverzeichnis Klappentext |
| Physical Description: | XXVI, 520 S. Ill., graph. Darst. |
| ISBN: | 0817643419 0817643400 |
Staff View
MARC
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| 100 | 1 | |a Rosenberg, Ian M. |d 1949- |e Verfasser |0 (DE-588)130602272 |4 aut | |
| 245 | 1 | 0 | |a Protein analysis and purification |b benchtop techniques |c Ian M. Rosenberg |
| 250 | |a 2. ed. | ||
| 264 | 1 | |a Boston [u.a.] |b Birkhäuser |c 2005 | |
| 300 | |a XXVI, 520 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 650 | 7 | |a Proteínas (análise) |2 larpcal | |
| 650 | 4 | |a Protéines - Analyse - Manuels de laboratoire | |
| 650 | 4 | |a Protéines - Purification - Manuels de laboratoire | |
| 650 | 4 | |a Proteins |x Analysis |v Laboratory manuals | |
| 650 | 4 | |a Proteins |x Purification |v Laboratory manuals | |
| 650 | 4 | |a Proteins |x analysis |v Laboratory Manuals | |
| 650 | 4 | |a Proteins |x isolation & purification |v Laboratory Manuals | |
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Record in the Search Index
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| adam_text | Contents
Preface
Acknowledgments
Chapter
Introduction
Kits, Cores, and Computers
How to Use This Book
Basic Laboratory Equipment
Laboratory Automation
Beyond Protein Analysis and Purification
Chapter
Introduction
A. The
B. The Four Levels of Protein Structure
Primary Structure
Secondary Structure
Tertiary Structure
Quaternary Structure
С
Hydrophobicity
Consensus Sequences
Proteomics
©
ity are followed. Take precautions when disposing of radioactive waste. Refer to Appendix A for further safety tips.
® Toxic substances are used. Exercise caution when handling. Refer to Appendix A for further safety rips.
xii Contents
Chapter
Introduction
A. Labeling Cells and Proteins
Metabolic Labeling Cells in Culture
©
® Protocol
® Protocol
Labeling Proteins Present at the Plasma Membrane
©
©
Protocol
©
Precipitation
©
B. Lysis
Lysis Buffers
©
Protocol
©
C. Principles of Immunoprecipitation
Antibodies as Detection Tools
Polyclonal Antibodies
Monoclonal Antibodies
Antibody Based Analytical Techniques: Western Blotting and
Immunoprecipitation
® Protocol
Protein Interaction Analysis
® Protocol
Reimmunoprecipitation of Immune Complexes
® Protocol
IP-Western Detection: Analysis of the Immunoprecipitate under
Non-Reducing Conditions
® Protocol
D. Additional Methods to Identify Associated Proteins
Sucrose Gradients
Protocol
Fractionating a Sucrose Gradient
Chemical Cross-Linking
General Considerations for Cross-Linking
Protocol
Receptor-Iigand Interaction
Protocol
® Protocol
Contents xiii
®
Homobifunctional Reagent Dithiobis (succinimidyl propionate) (DSP)
Analysis of Protein-Protein Interactions
Mapping Protein-Protein Contact Sites
Yeast Two-Hybrid Systems
Analyzing Protein Interactions by Fluorescence Resonance Energy
Transfer (FRET)
Chapter
Introduction to Polyacrylamide Gel Electrophoresis (PAGE)
A. Preparation of SDS-Polyacrylamide Gels
Protocol
Choosing the Acrylamide Concentration
® Protocol
® Protocol
® Protocol
Protocol
Protocol
Apparatus and Loading the Samples
Protocol
® Protocol
Tricine-SDS-PAGE (TSDS-PAGE)
Safety Considerations
B. 2-Dimensional (2-D) Gel Systems
Isoelectric Focusing
® Protocol
Single-Step Extraction/Solubilization Buffer
©
Gels
® Protocol
Flaws with 2-D Analysis
©
® Protocol
(NEPHGE)
Protocol
Fluorescence Two-Dimensional Difference Gel Electrophoresis
(2-D DIGE)
Protocol
2-D PAGE Databases
Protocol
xiv Contents
C.
Protocol
Protocol
Protocol
Viewing and Imaging a SYPRO Ruby-Stained 1-D or 2-D Gel
Protocol
Protocol
Imidazole and Zinc Salts
Protocol
D. Recovery of Proteins from the Gel
©
Protocol
E. Identification of Enzyme Activity in Polyacrylamide Gels
General Considerations
® Protocol
in the Separating Gel
® Protocol
Zymography
Protocol
Substrate Solution after Electrophoresis
Protocol
Polyacrylamide Gels
Identification of
® Protocol
Chapter
Introduction
A. Making a Cell Free Extract
Cellular Disruption
Extraction Buffer Composition
Protease Inhibitors
Methods of Cell Disruption
Clarification of the Extract
Protocol
Protocol
Protocol
Subcellular Markers
B. Protein Quantitation
The Bradford Method
® Protocol
Contents xv
Protocol
Protocol
Compatible Substances for the BCA Protein Assay
Incompatible Substances
Protocol
Based Assay of Proteins in Solution
С
Stabilization and Storage of Proteins
Concentrating Proteins from Dilute Solutions
Protocol
Precipitation
Ultrafiltration
Lyophilization
Dialysis
Protocol
Changing the Buffer by Gel Filtration
D. Precipitation Techniques
Protocol
Protocol
Precipitation with Polyethylene
Protocol
Protocol
Precipitation by Selective Denaturation
Protocol
Chloroform Precipitation
Protocol
Precipitation
Protocol
What to Do When All Activity Is Lost
Chapter
Introduction
A. Peripheral Membrane Proteins
Protocol
Protocol
B. Integral Membrane Proteins
Organic Alcohol Extraction of Peripheral Membrane Proteins
xvi Contents
Protocol
Protocol
С.
Properties of Detergents
Critical Micelle Concentration (CMC)
Micelle Molecular Weight
Hydrophile-Lipophile Balance (HLB)
Classification of Detergents
Ionic Detergents
Nonionic Detergents
Bile Salts
Detergent Solubilization
Choosing a Detergent
Choice of Initial Conditions
Protocol
©
Protein-to-Detergent Ratio
Detergent Removal
Removal of Ionic Detergents
Removal of Nonionic Detergents
Extracti-Gel® D
Chapter
Membrane Supports
Introduction
A. Transfer of Proteins to Membrane Supports
Protocol
Polyvinylidene Difluoride
Troubleshooting Western Blots
Protocol
Polypeptides on Membranes in the Presence of ZnCl2
Protocol
Protocol
B. Staining the Blot
Protocol
Protocol
Protocol
Brilliant Blue
Protocol
Acid/Schiff (PAS)
Contents xvii
C.
Protocol
Protocol
System
Protocol
Protocol
Ligand Blotting
Southwestern Blotting
Far Western Blotting
® Protocol
Protocol
Protocol
D. Detection of the Target Protein
Protein A
Second Antibody Conjugate
Biotin Avidin System
Protocol
Protocol
from Immunoblots
Enzymatic Detection Methods
Horseradish Peroxidase
® Protocol
3,3 ,4,4ЧеггаатіпоЬіргіепу1)
Protocol
Protocol
Protocol
Detection of
©
Removing Unwanted Background Signal from X-ray Film
©
Chapter
Introduction
A.
Protocol
Preparing the Target Protein for Digestion
B. Enzymatic Cleavage of Proteins
® Protocol
Electrophoresis
Cleavage of Proteins Transferred to PVDF or NC Membranes
Protocol
xviii Contents
Protocol
PVDF Membrane
C. Chemical Cleavage
® Protocol
PVDF Membrane —
©
Protocol
Polyacrylamide Gels
Protocol
® Protocol
D. Microsequencing from PVDF Membranes
When, and In What Form Do You Submit the Target Protein to the
Protein Sequencing Core?
® Protocol
PVDF Membranes
Sequencing Glycopeptides
® Protocol
Target Protein
Identifying Proteins Using Mass Spectrometry
Preparation of Proteins for MS Analysis
Partial Proteolysis
©
® Protocol
Membrane Proteins
Elution of Target Protein from SDS-PAGE
Protein Transfer to a Membrane
Considerations
MS Basics
Electrospray and Tandem Mass Spectrometry
MALDI and Peptide-Mass Mapping
Post-Source Decay (PSD) MALDI-MS
Protein Identification by MS
Peptide
Peptide
Peptide
Peptide
Identification of a Gene Product
Posttranslational Modifications and MS
Caveats When Using MS
Protein Database Searches
Bioinformatics
Contents xix
The Rise of Biological Databases
Search Engines
Databases
Identifying a Target Protein
Gene Analysis Tools
Subcellular Localization
Protein Domain Families
Structural Classification of Proteins
Genomic Organization
Additional Useful Sites on the Internet
PCR Primer Design Programs
Microarrays and Data Mining
Chapter
Modifications
Introduction
A. Glycosylation
Protocol
Acid (TFMS)
N-Glycosylation
Protocol
with N-Gly
Protocol
in Immunoprecipitates
® ® Protocol
O-Glycosylation
Protocol
Alkaline
Protocol
Immobilized on Blots
Protocol
Protocol
Combined Use of N-Glycanase and O-Glycanase
Protocol
Neuraminidase (NA)
Protocol
Neuraminidase
® Protocol
Neuraminidase
Lectins as Tools for Carbohydrate Analysis
Proteoglycans
©
xx Contents
B.
®
® Protocol
Protocol
Immunoblotting with Anti-Phosphoamino Acid Antibodies
©
[y-32P]GTP
Enzymatic Dephosphorylation
Protocol
Protocol
® Protocol
® Protocol
PVDF Membranes
® Protocol
Phosphopepetide and Phosphoamino Acid Analysis
® Protocol
® Two-Dimensional Phosphopeptide Mapping
® Protocol
for Phosphopeptide Mapping
Protocol
Protocol
in the First Dimension
Protocol
Protocol
TLC
® Protocol
® Protocol
from PVDF Membranes
® Protocol
Heat Treatment
® Protocol
® Protocol
and NaOH
® Protocol
C.
Palmitoylation and N-Myristoylation of Proteins
Analysis of Bound Fatty Acids
® Protocol
Proteins
Isoprenylation
® Protocol
Derivatives
® Protocol
Glypiation
Is the Target Protein Glycosyl Phosphatidylinositol Anchored?
Use of Triton X-114
Contents xxi
Protocol
Protocol
Triton X-114
Protocol
С
Metabolic Labeling with Precursors of the GPI Structure
Use of Anti-CRD
D. Selected Modifications
Transamidation
Acetylation
Methylation
Hydroxylation of
Degradation
Ubiquitination
Proteolytic Processing
Chapter
Introduction
A. Important Terminology Used in Chromatography
B. Gel Filtration Chromatography
Choice of Buffer
Choice of Column Size
Protocol
Protocol
How Rate
Hydrostatic Pressure
Sample Application
Protocol
Loading Sample Under the Eluent
Making Sure the Column Does Not Run Dry
Molecular Weight Determination
Spin Columns Used in Gel Filtration
Protocol
Protocol
С
Packing Materials
Column Designs
Column Guards
Detectors
xxii Contents
Choosing the Right Conditions
HPLC—Size Exclusion
D. Ion Exchange Chromatography: Separation on the Basis of Charge
Simplified Theory of Ion Exchange
Functional Groups on Exchange Columns
Choice of Exchanger Matrix
Preparation of the Exchanger
Choice of Buffer
Batch Adsorption
Protocol
Protocol
Experimental Tips
Elution-Step or Linear Gradient?
Protocol
Protocol
Protocol
Removing the Polybuffer
HPLC-Ion Exchange Chromatography
Membrane Adsorbers
Perfusion
E.
Simplified Theory of
Protocol
Protocol
Reversed Phase HPLC
Reversed Phase HPLC for the Isolation of Peptides
Multidimensional Liquid Chromatography
F. Affinity Chromatography
Immunoaffinity Purification
Protocol
Protocol
Protocol
Flow Rate
Binding Antigens to Immunoaffinity Matrices
Nonspecific Interactions
Protocol
Elution of Antigens from Immunoaffinity Matrices
Protocol
Ligand Affinity Chromatography
Contents xxiii
Protocol
Ester Derivatives of
Toluene Sulfonyl Chloride (Tosyl Chloride)
Pseudo-Affinity Adsorbents
Lectin Affinity Chromatography
Protocol
Agglutinin (WGA)
Protocol
Chromatography
Protocol
Fusion Protein
Protocol
Chapter
Introduction
Recombinant
Protein for Biochemical or Cell Biological Studies
A. In vitro Transcription and Translation
Protocol
Protocol
Protocol
Cap to mRNA
® Protocol
® Protocol
Microsomal Membranes
Protocol
Protease Digestion
Protocol
Endoglycosidase
Protein Transduction: A Method for Introducing Exogenous
Proteins into Cells
B.
Characterization
Expression and Purification of lacZ and trpE Fusion Proteins
Protocol
Protocol
Protocol
Protocol
Protocol
Bodies in
C.
Removing the Tag
xxiv Contents
Glutathione-S-Transferase
Protocol
Transformants
Protocol
Protocol
Protocol
Maltose Binding Protein
Staphylococcal Protein A and ZZ
Green Fluorescent Protein (GFP)
D. Expression of Foreign Proteins in Eukaryotic Cells
Expression and Isolation of
Protocol
Expression of Proteins in Insect Cells Using Baculoviral Vectors
Expression of Foreign Proteins in Mammalian Cells
Transfection: Expression of
in Eukaryotic Systems
Protocol
Calcium Phosphate
Protocol
Protocol
Protocol
Protocol
Appendices
A. Safety Considerations
First Aid: Emergency Procedures
B. Antibody Preparation
Production of Polyclonal
Protocol B.I Preparation of the Antigen-Adjuvant Emulsion
Protocol B.2 Intramuscular Immunization
Protocol B.3
Protocol B.4 Subcutaneous Immunization
Protocol B.5 Bleeding the Rabbit and Serum Preparation
Protocol B.6 Precipitation of IgG with Saturated Ammonium
Sulfate — 438
Purification of Antibody Using Protein A Affinity Columns
Protocol B.7 Purifying Total
Numbering Mice
C. Solutions
Commercial Strengths of Common Laboratory Chemicals
Water
Contents xxv
Molarity..................................................... 442
Choosing and Preparing Buffers
Common Laboratory Solutions
Extinction Coefficients
D. Nucleic Acids
Spectrophotometric Conversions
DNA/Protein Conversions
Oligonucleotide Concentrations
Protocol D.I Fluorometric Estimation of
RNA
E. Modifications and Motifs
Nomenclature
Protein Modification Sequences
Protein Kinase Recognition Sequence Motifs
Subcellular Localization Motifs
Protein Databases
F. Centrifugation
Nomogram
General Purpose Centrifuge Rotors
Ultracentrifuge
G. Proteases and Proteolytic Enzyme Inhibitors
Commonly Used Proteases
Protocol G.I Preparation of Defatted BSA
Protease Inhibitors
H. Radioactivity
Manual and Machine Film Processing
I. Tissue Culture
Transwell Permeable Supports
J. Miscellaneous
Protocol J.I Siliconizing Glassware
Unit Prefixes
The Greek Alphabet
Abbreviations
HPLC Pump Pressure Conversion
xxvi Contents
Dipeptide
Mass Differences Considered in Molecular Weight Analysis
of Proteins
K. List of Suppliers, Vendors, Manufacturers
Index
New technologies and the manner in which scientific research is
conducted have been fully incorporated into this thoroughly revised
second edition of PROTEIN ANALYSIS AND PURIFICATION. Among
the many topics introduced to this new edition are the use of robotics
in the lab, the impact of the codification of the human genome,
functional proteomics, mass spectrometry, and the expanding use of
bioinformatics as a tool to improve efficiency and productivity. The
author has also included an annotated appendix of the educational
materials and technical databases available online to enhance the
researcher s benchside access to the latest resources. With solid
explanations of the science asking the why and literally dozens of
step-by-step protocols providing the how, this new edition is a
complete resource for the benchside.
|
| any_adam_object | 1 |
| author | Rosenberg, Ian M. 1949- |
| author_GND | (DE-588)130602272 |
| author_facet | Rosenberg, Ian M. 1949- |
| author_role | aut |
| author_sort | Rosenberg, Ian M. 1949- |
| author_variant | i m r im imr |
| building | Verbundindex |
| bvnumber | BV019439082 |
| callnumber-first | Q - Science |
| callnumber-label | QP551 |
| callnumber-raw | QP551 |
| callnumber-search | QP551 |
| callnumber-sort | QP 3551 |
| callnumber-subject | QP - Physiology |
| classification_rvk | VK 8560 WC 4170 |
| classification_tum | CHE 820f CHE 810f CHE 808f |
| ctrlnum | (OCoLC)55067931 (DE-599)BVBBV019439082 |
| dewey-full | 572/.6 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572/.6 |
| dewey-search | 572/.6 |
| dewey-sort | 3572 16 |
| dewey-tens | 570 - Biology |
| discipline | Chemie / Pharmazie Biologie Chemie |
| edition | 2. ed. |
| format | Book |
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| id | DE-604.BV019439082 |
| illustrated | Illustrated |
| indexdate | 2024-07-09T20:00:11Z |
| institution | BVB |
| isbn | 0817643419 0817643400 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-012892618 |
| oclc_num | 55067931 |
| open_access_boolean | |
| owner | DE-20 DE-91G DE-BY-TUM DE-703 DE-M49 DE-BY-TUM DE-355 DE-BY-UBR DE-19 DE-BY-UBM DE-11 DE-188 |
| owner_facet | DE-20 DE-91G DE-BY-TUM DE-703 DE-M49 DE-BY-TUM DE-355 DE-BY-UBR DE-19 DE-BY-UBM DE-11 DE-188 |
| physical | XXVI, 520 S. Ill., graph. Darst. |
| publishDate | 2005 |
| publishDateSearch | 2005 |
| publishDateSort | 2005 |
| publisher | Birkhäuser |
| record_format | marc |
| spelling | Rosenberg, Ian M. 1949- Verfasser (DE-588)130602272 aut Protein analysis and purification benchtop techniques Ian M. Rosenberg 2. ed. Boston [u.a.] Birkhäuser 2005 XXVI, 520 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Proteínas (análise) larpcal Protéines - Analyse - Manuels de laboratoire Protéines - Purification - Manuels de laboratoire Proteins Analysis Laboratory manuals Proteins Purification Laboratory manuals Proteins analysis Laboratory Manuals Proteins isolation & purification Laboratory Manuals Sequenzanalyse Chemie (DE-588)4132277-0 gnd rswk-swf Proteine (DE-588)4076388-2 gnd rswk-swf Chemische Analyse (DE-588)4009840-0 gnd rswk-swf Trennverfahren (DE-588)4078395-9 gnd rswk-swf Proteine (DE-588)4076388-2 s Chemische Analyse (DE-588)4009840-0 s DE-604 Trennverfahren (DE-588)4078395-9 s Sequenzanalyse Chemie (DE-588)4132277-0 s 1\p DE-604 Digitalisierung UB Regensburg application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=012892618&sequence=000003&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis Digitalisierung UB Regensburg application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=012892618&sequence=000004&line_number=0002&func_code=DB_RECORDS&service_type=MEDIA Klappentext 1\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk |
| spellingShingle | Rosenberg, Ian M. 1949- Protein analysis and purification benchtop techniques Proteínas (análise) larpcal Protéines - Analyse - Manuels de laboratoire Protéines - Purification - Manuels de laboratoire Proteins Analysis Laboratory manuals Proteins Purification Laboratory manuals Proteins analysis Laboratory Manuals Proteins isolation & purification Laboratory Manuals Sequenzanalyse Chemie (DE-588)4132277-0 gnd Proteine (DE-588)4076388-2 gnd Chemische Analyse (DE-588)4009840-0 gnd Trennverfahren (DE-588)4078395-9 gnd |
| subject_GND | (DE-588)4132277-0 (DE-588)4076388-2 (DE-588)4009840-0 (DE-588)4078395-9 |
| title | Protein analysis and purification benchtop techniques |
| title_auth | Protein analysis and purification benchtop techniques |
| title_exact_search | Protein analysis and purification benchtop techniques |
| title_full | Protein analysis and purification benchtop techniques Ian M. Rosenberg |
| title_fullStr | Protein analysis and purification benchtop techniques Ian M. Rosenberg |
| title_full_unstemmed | Protein analysis and purification benchtop techniques Ian M. Rosenberg |
| title_short | Protein analysis and purification |
| title_sort | protein analysis and purification benchtop techniques |
| title_sub | benchtop techniques |
| topic | Proteínas (análise) larpcal Protéines - Analyse - Manuels de laboratoire Protéines - Purification - Manuels de laboratoire Proteins Analysis Laboratory manuals Proteins Purification Laboratory manuals Proteins analysis Laboratory Manuals Proteins isolation & purification Laboratory Manuals Sequenzanalyse Chemie (DE-588)4132277-0 gnd Proteine (DE-588)4076388-2 gnd Chemische Analyse (DE-588)4009840-0 gnd Trennverfahren (DE-588)4078395-9 gnd |
| topic_facet | Proteínas (análise) Protéines - Analyse - Manuels de laboratoire Protéines - Purification - Manuels de laboratoire Proteins Analysis Laboratory manuals Proteins Purification Laboratory manuals Proteins analysis Laboratory Manuals Proteins isolation & purification Laboratory Manuals Sequenzanalyse Chemie Proteine Chemische Analyse Trennverfahren |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=012892618&sequence=000003&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=012892618&sequence=000004&line_number=0002&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT rosenbergianm proteinanalysisandpurificationbenchtoptechniques |