细胞生物学实验：蛋白质（导读版） chm 下载 lrf pdf 阿里云 azw3 kindle 网盘

随着实验室技术的流水线化和生物学研究方法的不断发展，对跨学科实验方法的理解与掌握成为现代生物学家的素质。作为CellBiology，ThirdEdition的选编版，本书提供了多种有关蛋白质的实验方法：目的蛋白质的鉴定、蛋白质的检测和分析、蛋白质-蛋白质互作与DNA-蛋白质互作的鉴定。这本实验手册提供了详细的实验步骤，强调生物学问题并讨论每项技术的细节以提供说明，适用于蛋白质研究领域的初学者和具备一定实验经验的研究者。

前言

撰稿人名单

Ⅰ 标记和检测蛋白质

    1.蛋白质确定

    2.磷酸肽作图:一个基本方案

    3.蛋白质和肽的放射性碘化

    4.银染法检测凝胶中的蛋白质:一种与质谱分析兼容的方法

    5.用SYPRO染料荧光检测凝胶中的蛋白质

    6.放射自显影法和荧光自显影法:基于胶片的平面标本放射性成像技术

Ⅱ 基于蛋白质分离的蛋白质组学分析

    7.自由流电泳

    8.基于凝胶的蛋白质组学:蛋白质的高分辨双向凝胶电泳。等电聚焦和非平衡pH梯度电泳

    9.高分辨双向电泳结合固定化pH梯度的蛋白质组学分析

    10.双向差异凝胶电泳:应用于多生物样本中蛋白表达差异分析

    11.图像分析和定量

    12.用体内同位素标记方法对翻译后修饰蛋白质进行双向凝胶分析

Ⅲ 蛋白质-蛋白质相互作用和蛋白质-小分子相互作用

    13.非变性条件下蛋白质的免疫沉淀

14.非变性聚丙烯酰胺凝胶电泳作为研究蛋白质相互作用的一种方法:在线粒体氧化磷酸化复合物分析中的应用

    15.化学交联法分析蛋白质-蛋白质相互作用

    16.靶向过氧化物酶体作为检测蛋白质-蛋白质相互作用的工具

    17.生物分子相互作用分析质谱

    18.配体印迹重叠实验:检测钙离子和小GTP结合蛋白

    19.模块规模酵母双杂交筛选

20.亲和电泳研究生物特异的相互作用:高分辨双向亲和电泳用于从小鼠血浆中将半抗原特异的多克隆抗体分离至单克隆抗体

    21.蛋白质泛素化研究方法

Ⅳ 蛋白质-DNA相互作用

    22.凝胶迁移率改变分析

    23.转录因子的DNA亲和层析:寡核苷酸捕获技术

Ⅴ 用于鉴定蛋白质及其修饰的质谱方法

    24.蛋白质鉴定和序列测定的质谱方法

    25.蛋白质组特异的样品制备方法用于基质辅助激光解析/离子化质谱分析

    26.蛋白质点的胶内酶切用于质谱分析

    27.用串联质谱法进行肽序列测定

    28.用肽的串联质谱谱图直接进行数据库检索

    29.运用串联质谱法和序列相似性数据库搜索工具对未测序基因组生物的蛋白质进行鉴定

    30.运用质谱鉴定蛋白质磷酸化位点

    31.运用质谱进行糖类或糖蛋白分析

    32.稳定同位素标记细胞培养中氨基酸用于定量蛋白质组学

    33.在复杂多肽混合物中对半胱氨酸肽进行位点特异性的稳定同位素标记

    34.用电喷雾离子化质谱测量蛋白质氢交换

    35.非凝胶蛋白质组学:选择性反向色谱从复杂多肽混合物中分离含甲硫氨酸的肽

    36.质谱法研究非共价的蛋白质相互作用和蛋白质装配体

索引

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SECTIONI

LABELING AND DETECTING

PROTEINS

CHAPTER 1

Protein Determination

Martin Guttenberger

I.Introduction 3

II.Materials and Instrumentation 3

A.Lowry Assay 4

B.Bradford Assay 4

C.Neuhoff Assay (Dot-Blot Assay) 4

III.Procedures 4

A.Lowry Assay 5

B.Bradford Assay 5

C.Dot-Blot Assay 6

IV.Comments 8

A.Lowry Assay 9

B.Bradford Assay 9

C.Dot-Blot Assay 9

V.Pitfalls 10

A.Lowry Assay 10

B.Bradford Assay 11

C.Dot-Blot Assay 11

References 12

I.INTRODUCTION

The protein content of tissues or samples can

serve a number of purposes:It can be a research

topic of its own (e.g.,in nutritional studies;

Hoffmann et al.,2002),a loading control in gel

electrophoresis (ünlü et al.,1997),or a reference

quantity in biochemical (e.g.,yields in protein

purification) or physiological (e.g.,specific activities

of enzyme preparations; Guttenberger et al.,

1994) investigations.In addition,with the advent

of proteomics,there is an increasing need for

protein quantitation in complex sample buffers

containing detergents and urea as potentially

interfering compounds (ünlü et al.,1997).In

any case,care should be taken to obtain correct

results.This article focuses on three techniques

and outlines the specific pros and cons.

II.MATERIALS AND

INSTRUMENTATION

The following reagents are from the indicated

suppliers.All other reagents are of analytical

grade (Merck):

A.Lowry Assay

From Lowry et al.(1951):Folin–Ciocalteu phenol

reagent (Merck,Cat.No.1.09001).A detergentcompatible

modification of the Lowry assay is

available as a kit (Bio-Rad 500-0116).

B.Bradford Assay

From Bradford (1976):Coomassie brilliant

blue G-250 (Serva Blue G,Serva,Cat.No.35050).

The reagent for this assay is available commercially

from Bio-Rad (Cat.No.500-0006).

C.Neuhoff Assay (Dot-Blot Assay)

From Guttenberger et al.(1991) and Neuhoff

et al.(1979):Ammonium sulfate for biochemical

purposes (Merck,Cat.No.1.01211),benzoxanthene

yellow (Hoechst 2495,Merck Biosciences,

Cat.No.382057,available upon request),cellulose

acetate membranes (Sartorius,Cat.No.SM

11200),glycine,and SDS (Serva,Cat.Nos.23390

and 20763,respectively).Commercially available

ammonium sulfate frequently contains substantial

amounts of undefined UV-absorbing and fluorescing

substances.These lead to more or less

yellowish solutions.Use only colourless solutions

to avoid possible interference in fluorometry.

Solutions are prepared from bidistilled water.

Bovine serum albumin (BSA,fraction V,Roche,

Cat.No.735086) is used as a standard protein.

Ninety-six-well,flat-bottomed polystyrene microtiter

plates (Greiner,Cat.No.655101) are used for

the photometric tests.

III.PROCEDURES

With respect to convenience and speed,

microplate reader assays are described where

appropriate.These assays can be read easily in

conventional instruments by employing microcuvettes

or by scaling up the volumes (fivefold).

The composition of the sample (extraction)

buffer requires thought with respect to the

avo idance of artifactual alterations of the protein

and to the compatibility with the intended

experimental procedures.The former requires

strict control of adverse enzyme activities (especially

proteases and phenol oxidases) and,in

the case of plant tissues,of interactions with

secondary metabolites.A convenient,semiquantitative

assay for proteolytic activities allowing

for the screening of suitable inhibitors was

described by Gallagher et al.(1986).There is

some uncertainty as to which assay gives the

most reliable results in combination with extracts

from plant tissues rich in phenolic substances.

The influence of such substances can

never be predicted.It is therefore imperative to

minimize interaction of these substances with

protein in the course of sample preparation.For

a more detailed discussion of this problem,see

Guttenberger et al.(1994).

A frequent source of ambiguity is the use of

the term “soluble protein.” Soluble as opposed to

membrane-bound proteins stay in solution during

centrifugation for 1 h at 105,000 g (Hjelmeland

and Chrambach,1984).

All assays described in this article quantitate

protein relative to a standard protein.The

choice of the standard protein can markedly

influence the result.This requires special attention

for proteins with a high content of certain

amino acids (e.g.,aromatic,acidic,or basic

amino acids).For most accurate results,choose

a standard protein with similar amino acid

composition or,if not available,compare different

assays and standard proteins.Alternatively,

employ a modified Lowry procedure that

allows for absolute quantitation of protein

(Raghupathi and Diwan,1994).

The most efficient way to prepare an exact

dilution series of the standard protein employs

a handheld dispenser (e.g.,Eppendorf multipette).

Typically a six-point series is pipetted

according to Table 1.1.In any case,avoid

a concentration gradient of the sample buffer.

Usually samples and standards may be kept

at -20℃ for a couple of weeks.For longer storage

intervals,keep at -80℃.

A.Lowry Assay

See Lowry et al.(1951).

Solutions

Note:For samples low in protein (0.02 mg·ml-1

or less),prepare reagents A and B at double

strength.

1.Reagent A:2% (w/v) sodium carbonate

(Na2CO3) in 0.10N NaOH.To make 1 litre

of reagent A (5000 determinations),dissolve

20 g Na2CO3 in 1 litre 0.10 M NaOH.Keep at

room temperature in tightly closed screwcap

plastic bottles.

2.Reagent B:0.5% CuSO4·5 H2O in 1% sodium

or potassium tartrate.To make 20 ml of

reagent B,dissolve 0.1 g CuSO4·5 H2O in

20 ml 1% tartrate (0.2 g sodium or potassium

tartrate dissolved in 20 ml water).Keep at

room temperature.

3.Reagent C (alkaline copper solution):Mix 25 ml

of reagent A and 0.5 ml of reagent B.Prepare

fresh each day.

4.Reagent D (Folin–Ciocalteu phenol reagent):

Dilute with an equal volume of water just

prior to use

Steps

1.Place 4 ul of sample (protein concentration

0.02–1 mg·ml-1) or blank into cavities of a

microplate or into appropriate test tubes.

2.Add 200 ul of reagent C and mix.Allow to

stand for at least 10 min.

3.Add 20 ul of reagent D and mix immediately.

Allow to stand for 30 min or longer.

4.Read the samples in a microplate reader or

any other photometer at 750 nm.

Modifications

1.The sample volume may be raised to

140 ul when samples are low in protein

(0.02 mg·ml-1 or less).In this case,employ

double-strength reagent C.

2.If samples have been dissolved in 0.5 M

NaOH (recommended for resolubilization

of acid precipitates),omit NaOH from

reagent A.

B.Bradford Assay

See Bradford (1976).

Solutions

1.Protein reagent stock solution:0.05% (w/v)

Coomassie brilliant blue G-250,23.8%

(v/v) ethanol,42.5% (w/v) phosphoric

acid.To make 200 ml of stock solution

(5000 determinations),dissolve 0.1 g Serva

blue G in 50 ml 95% ethanol (denatured

ethanol works as well),add 100 ml 85%

phosphoric acid,and make up to 200 ml by

adding water.The stock solution is available

commercially (Bio-Rad).Keep at 4℃.The

reagent contains phosphoric acid and

ethanol or methanol.Handle with due care

(especially when employing a dispenser)!

2.Protein reagent:Prepare from the stock

solution by diluting in water (1:5).Filter

immediately prior to use.

Steps

1.Place 4 ul of sample (protein concentration

0.1–1 mg·ml-1) or blank into cavities of a

microplate or into appropriate test tubes.

2.Add 200 ul of protein reagent and mix.

Allow to stand for at least 5 min.

3.Read the samples within 1 h in a microplate

reader or any other photometer at 595 nm.

Modifications

1.For improved linearity and sensitivity,

compute the ratio of the absorbances,590 nm

over 450 nm (Zor and Selinger,1996).

2.Microassay:For diluted samples (less than

0.1 mg·ml-1),proceed as follows:Employ

200 ul of sample and add 50 ul of protein

reagent stock.

C.Dot-Blot Assay

See Guttenberger et al.(1991).Do not change

the chemistry of the membranes.Nitrocellulose

will dissolve in the staining solution; PVDF

membranes develop a strong background.

Solutions

1.Benzoxanthene stock:To prepare the stock

solution add 1 ml of water to 0.5 g of the

fluorescent dye (as supplied,weighing not

necessary); keep at -20℃.The toxicity of

benzoxanthene is not thoroughly studied,it

might be mutagenic!

2.Destaining solution:Methanol/acetic acid

(90/10,v/v).To make 1 litre,mix 100 ml

acetic acid and 900 ml methanol.

3.Staining solution:To obtain 100 ml,dilute 80 ul

benzoxanthene stock in 100 ml destaining

solution.Be sure to pour the destaining

solution onto the stock solution to prevent

the latter from clotting.Keep staining and

destaining solutions in tightly closed screwcap

bottles at 4℃ in the dark.They are stable

for months and can be used repeatedly.Take

due care in handling the highly volatile

solutions containing methanol!

4.SDS stock:To make 30 ml of 10% (w/v)

SDS stock solution,dissolve 3 g SDS in

approximately 20 ml of water,stir,and make

up to 30 ml (allow some time for settling of

foam).Keep at room temperature; it is stable

for at least 1 year.

5.Elution buffer:0.25 M glycine–sulfuric acid

buffer (pH 3.6) and 0.02% (w/v) SDS.To

prepare 1 litre,dissolve 18.8 g glycine in

approximately 900 ml water and add 15 ml

of 0.5 M sulfuric acid.Slight deviations

from pH 3.6 are tolerable.Add 2 ml SDS

stock and make up to 1 litre.Keep at room

temperature; it is stable for months.

The following solutions are not needed for

the standard protocol.

6.Washing solution A:Saturated ammonium

sulfate,adjust to pH 7.0 with Tris.To make 1

litre,stir ammonium sulfate in warm water

(do not heat excessively).Let the solution cool

to room temperature overnight and titrate to

pH 7.0 with a concentrated (approximately

2 M) solution of Tris (usually approximately

1 ml is required).Keep at room temperature.

As ammonium sulfate tends to produce

lumps in the storage bottle it might be easier

to weigh the entire bottle,add some water,

remove the resulting slurry,and weigh the

empty bottle again.To produce a saturated

solution (53.1%,w/v),dissolve 760 g

ammonium sulfate in 1 litre water.

7.Washing solution B:Methanol/acetic acid/

water (50/10/40,v/v).To make 1 litre,mix

100 ml acetic acid and 500 ml methanol;

make up to 1 litre.Keep at 4℃.

8.Drying solution:1-Butanol/methanol/acetic

acid (60/30/10,v/v).To make 0.1 litre,mix

10 ml acetic acid,30 ml methanol,and 60 ml

butanol.Keep at 4℃; use up to six times.

Steps

The dot-blot assay is a versatile tool; its different

modifications enable one to cope with

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书籍介绍

《细胞生物学实验——蛋白质(英文版)》的目录和前言已经译成中文，正文部分保留英文原版。另附中国医学科学院基础医学研究所博士生导师高友鹤教授所作精彩导读一篇。

随着实验室技术的简化和生物学研究方法的扩展，更好地了解跨学科方法已经成为现代生物学家的必备优点。《细胞生物学实验——蛋白质》一书包含了《细胞生物学实验手册》(第三版)中的选集，提供了用于鉴定感兴趣的蛋白质、检测和分析这些蛋白质，确定蛋白质-蛋白质相互作用以及DNA-蛋白质相互作用的各种方法。

这本循序渐进的实验手册解决生物学问题并且通过详细讨论对每种技术提供指导，使其对于初学者和经验丰富的研究人员而言都是一本重要的指南。