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该书有超过 80 页的实验方案和疑难解答提示，并配有全彩插图描述如何最佳使用Abcam的抗体产品

Abcam的在线实验方案很受欢迎，因此Abcam制作了印刷版本便于实验室使用，它覆盖了最常用的实验室应用，包括：

荧光
蛋白质印迹
免疫组织化学
流式细胞术
ELISA
免疫沉淀反应
ChIP
缓冲液
母液

该书专为任何需要这些技术的已测试实验方案的实验人员设计。该书还包括抗体选择信息和保存指南。

目录
欢迎来到 Abcam
关于 Abcam .......................................................................... 1
Abcam 帐户的好处 ............................................................... 2
Abreviews® and Abpoints® ................................................... 2
技术资料
第一章：抗体和抗体的结构 .................................................. 3
第二章：抗体纯化 ................................................................ 6
第三章：抗体选择和稀释 ..................................................... 7
第四章：荧光 ....................................................................... 9
4.1 荧光染色-工作原理 ........................................................ 9
4.2 荧光染料表 ................................................................... 11
第五章：蛋白质免疫印迹技术 ............................................ 13
5.1 样品准备 ...................................................................... 15
抽提缓冲液 ................................................................... 15
蛋白酶和磷酸酶抑制剂 ................................................. 16
细胞培养物的裂解准备 ................................................. 16
组织抽提准备 ............................................................... 16
蛋白质含量的测定 ........................................................ 17
凝胶上样准备 ............................................................... 17
5.2 电泳 .............................................................................. 18
PAGE 凝胶准备 ............................................................ 18
阳性对照 ....................................................................... 19
分子量 markers ............................................................ 19
上样和跑胶 ................................................................... 19
使用对照 ....................................................................... 20
5.3 蛋白质的转移和染色 .................................................... 20
蛋白在凝胶中的显色 ..................................................... 20
转移 .............................................................................. 20
蛋白在膜上的显色：丽春红 .......................................... 22
膜封闭 .......................................................................... 22
加一抗孵育 ................................................................... 22
加二抗孵育 ................................................................... 22
显色 .............................................................................. 23
5.4 参考文献 ...................................................................... 23
5.5 常见问题的解决方法 .................................................... 24
第六章：免疫组织化学和免疫细胞化学 .............................. 27
6.1 IHC-石蜡技术说明 (IHC-P) ........................................... 27
6.1.1 优化 IHC-P 抗体 .................................................. 29
6.1.2 脱蜡 ..................................................................... 29
6.1.3 抗原修复 .............................................................. 30
6.1.4 免疫组织化学染色 ............................................... 33
6.1.5 文献 ..................................................................... 37
6.2 免疫组织化学冷冻切片切割技术 (IHC-Fr) .................... 37
6.3 免疫细胞化学 (ICC) ...................................................... 37
6.4 IHC/ICC的固定和通透 .................................................. 38
6.5 灌注固定法 ................................................................... 40
6.6 鼠对鼠染色法 ............................................................... 41
6.7 疑难解答 IHC/ICC ........................................................ 42
第七章：ELISA .................................................................. 44
7.1 间接 ELISA .................................................................. 44
7.2 直接 ELISA .................................................................. 46
7.3 双抗夹心 ELISA ........................................................... 48
7.4 疑难解答-ELISA ........................................................... 50
第八章：流式细胞术 ........................................................... 53
8.1 流式细胞术/FACS 的推荐对照 ..................................... 55
8.2 直接染色步骤 ............................................................... 56
8.3 间接染色步骤 ............................................................... 57
8.4 细胞内染色 ................................................................... 58
8.5 流式细胞术常见问题分析 ............................................. 61
第九章：免疫沉淀 (IP) ....................................................... 63
9.1 裂解缓冲液 ................................................................... 63
9.2 抗体到珠的交联 ............................................................ 67
9.3 用 IgM 抗体进行免疫沉淀 ............................................ 68
9.4 免疫沉淀常见问题分析 ................................................. 69
第十章：染色质免疫沉淀 ................................................... 71
10.1 交联染色质免疫沉淀 (X-ChIP) .................................. 71
10.2 ChIP 常见问题分析 .................................................... 76
第十一章：缓冲液和储备溶液 ............................................ 77
11.1 PBS, 三乙醇胺缓冲液和三乙醇胺-
吐温缓冲液的配制 ....................................................... 77
11.2 免疫印迹 ..................................................................... 77
11.3 免疫组化 (IHC) ........................................................... 79
11.4 酶联免疫吸附试验 ...................................................... 80
11.5 流式细胞术 ................................................................. 80
11.6 免疫沉淀 ..................................................................... 80
11.7 染色质免疫沉淀 .......................................................... 80
第十二章：抗体保存指南 ................................................... 82
技术支持
Abcam 公司在线资源 ......................................................... 84
产品信息指南.................................................................84
在线实验设计 ..................................................................... 85
在线实验设计海报 .............................................................. 85

Contents
Protocols
SECTION 1: Antibodies and antibody structure . . . . . . .3
SECTION 2: Various formats of antibody and . . . . . . . . .6
antibody purification
SECTION 3: Choosing an antibody and antibody dilution . . .7
SECTION 4: Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . .9
4.1 How single fluorochromes work . . . . . . . . . . . . . . . . . . .9
4.2 How tandem dyes work . . . . . . . . . . . . . . . . . . . . . . . . . .9
4.3 Fluorochrome table (excitation and emission wavelengths) . . .11
SECTION 5: Western blot . . . . . . . . . . . . . . . . . . . . . . . .13
5.1 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . .15
Lysis buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
Protease and phosphatase inhibitors . . . . . . . . . . . . . . .16
Preparation of lysate from cell culture . . . . . . . . . . . . . . .16
Preparation of lysate from tissues . . . . . . . . . . . . . . . . . .16
Determination of protein concentration . . . . . . . . . . . . . .17
Preparation of samples for loading into gels . . . . . . . . . .17
5.2 Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
Preparation of PAGE gels . . . . . . . . . . . . . . . . . . . . . . . .18
Positive controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
Molecular weight markers . . . . . . . . . . . . . . . . . . . . . . . .19
Loading samples and running the gel . . . . . . . . . . . . . . .19
Use of loading controls . . . . . . . . . . . . . . . . . . . . . . . . . .20
5.3 Transfer of proteins and staining . . . . . . . . . . . . . . .20
Visualization of proteins in gels . . . . . . . . . . . . . . . . . . . .20
Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
Visualization of proteins in membranes: Ponceau Red . . . . .22
Blocking the membrane . . . . . . . . . . . . . . . . . . . . . . . . . .22
Incubation with the primary antibody . . . . . . . . . . . . . . . .22
Incubation with the secondary antibody . . . . . . . . . . . . .22
Development methods . . . . . . . . . . . . . . . . . . . . . . . . . . .23
5.4 Western blot references . . . . . . . . . . . . . . . . . . . . . . .23
5.5 Troubleshooting tips – Western blotting . . . . . . . . .24
SECTION 6: Immunohistochemistry . . . . . . . . . . . . . . . .27
6.1 IHC-Paraffin embedded . . . . . . . . . . . . . . . . . . . . . . . . .27
6.1.1 Optimizing a new antibody for IHC-P . . . . . . . . . .29
6.1.2 Deparaffinization . . . . . . . . . . . . . . . . . . . . . . . . . .29
6.1.3 Antigen retrieval . . . . . . . . . . . . . . . . . . . . . . . . . . .30
6.1.4 Immunostaining and detection protocol . . . . . . . . .33
6.1.5 Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37
6.2 IHC-Frozen sections (IHC-Fr) . . . . . . . . . . . . . . . . . . . .37
6.3 Immunocytochemistry (ICC) . . . . . . . . . . . . . . . . . . . . .37
6.4 IHC and ICC fixation and permeabilization tips . . . . . .38
6.5 Perfusion fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40
6.6 Mouse on mouse staining tips . . . . . . . . . . . . . . . . . . . .41
6.7 Troubleshooting tips – IHC and ICC . . . . . . . . . . . . . . .42
SECTION 7: ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44
7.1 Indirect ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44
7.2 Direct ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
7.3 Sandwich ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48
7.4 Troubleshooting tips - ELISA . . . . . . . . . . . . . . . . . . . . .50
SECTION 8: Flow Cytometry (introduction) . . . . . . . . . .53
8.1 Recommended controls for flow cytometry/FACs . . . .55
8.2 Direct Flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . .56
8.3 Indirect Flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . .57
8.4 Intracellular flow cytometry . . . . . . . . . . . . . . . . . . . . . .58
8.5 Flow cytometry troubleshooting . . . . . . . . . . . . . . . . . .61
SECTION 9: Immunoprecipitation . . . . . . . . . . . . . . . . . .63
9.1 Lysis buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63
9.2 Crosslinking antibody to the beads . . . . . . . . . . . . . . .67
9.3 Using IgM antibodies for IP . . . . . . . . . . . . . . . . . . . . .68
9.4 Troubleshooting tips - IP . . . . . . . . . . . . . . . . . . . . . . .69
SECTION 10: ChIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71
10.1 X-ChIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71
10.2 Troubleshooting tips - ChIP . . . . . . . . . . . . . . . . . . . .76
SECTION 11: Buffers and stock solutions . . . . . . . . . . .77
11.1 Standard PBS, TBS, TBS Tween . . . . . . . . . . . . . . . .77
11.2 Western blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77
11.3 IHC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79
11.4 ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .80
11.5 Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .80
11.6 IP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .80
11.7 ChIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .80
SECTION 12: Antibody storage guide . . . . . . . . . . . . . .82
Welcome to Abcam
About Abcam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Benefits of an Abcam account . . . . . . . . . . . . . . . . . . . . . . . .2
Abreviews® and Abpoints® . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Technical help
Online resources at Abcam . . . . . . . . . . . . . . . . . . . . . . . . .84
Product datasheet guide . . . . . . . . . . . . . . . . . . . . . . . . . . .84
Online protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85
Online poster library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85
Ordering and contact details
Contact details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .86
Local offices and distributors . . . . . . . . . . . . . . . . . . . . . . . .87
Required information for ordering . . . . . . . . . . . . . . . . . . . .88
Abcam’s terms and conditions . . . . . . . . . . . . . . . . . . . . . .89

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