WesternBolting实验
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Page 5
The Priciple of Western Blot
-
Page 7
1.Tissue preparation
-
SDS (sodium dodecyl sulfate十二烷基硫酸钠) is a detergent (清洁 剂)(soap) that can dissolve hydrophobic（ 难溶于水）molecules but also has a negative charge attached to it.
Towbin H, et al (1979). "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.". Proc Natl Acad Sci U S A. 76 (9): 4350–4354
Page 15
4. Fluorescent detection
results
Optimized antibody concentration, lower background
Primary Ab Secondary Ab
Page 16
1:1000 1:5000
1:1000 1:20000
the binding sites
Page 19
Medical Diagnosis
• • • HIV Lyme BSE ‘ mad cow disease’
The confirmatory HIV test
HIV has been found by ELISA To confirm the result
After ‘window period’
Page 21
Western Blot for Lyme
B. burgdorferi伯疏氏螺旋体（细胞外寄生菌） confirm the diagnosis
The Western blot IgM has a specificity of 94–96% for patients with clinical symptoms of early Lyme disease
Western Blot for Research Use
Identify the nature of the protein quantitative analysis of the micromolecule antigen Epitope ห้องสมุดไป่ตู้apping Identify the process of
Page 3
History/Background Northern Blot
Developed in 1977 by George Stark and his colleagues at Stanford University Study gene expression by detection of RNA (or isolated mRNA) in a sample
Page 8
Tissue preparation
Page 9
2.Gel electrophoresis
-
SDS-PAGE(SDS变性不连续聚丙烯酰胺凝胶电泳)
Page 10
3.Transfer
-
nitrocellulose（硝酸纤维薄膜） Tips: The difference between different membranes
Page 22
Bovine spongiform encephalopathy
mad cow disease
Piron
identified and differentiated the specific molecular weights
Page 23
Page 4
History/Background Western Blot (Immunoblotting)
George Stark developed an early protein blotting technique Harry Towbin`s faster and simpler approach is clearly preferred today Electrophoretic transfer of proteins from gels to membranes
1:1000 1:100000
The applications of Western Blot
— —3127119027 马欣俐
Scientific Research
• the protein is successfully expressed ？ • the target protein is present in a given cell? • the quantity of the protein?
Southern blot (DNA)
History/Background Southern Blot
‘Southern’ hybridization named after Sir Edwin Southern Developed in 1975 One of the most highly cited scientific publications Allow the detection of a specific DNA sequence (gene or other) in a large, complex sample of DNA (e.g. cellular DNA)
The History of Western Blotting
History/Background
Spawned naming of related techniques:
Northern blot (RNA)
Western blot (Protein)
Eastern blot
Page 2
Page 11
Tissue preparation
Page 12
4.Blocking
-
Tips: one step and two steps
Page 13
Tissue preparation
one step
two steps
Page 14
5.Analysis
-
1. Colorimetric detection 2. Chemiluminescent detection 3. Radioactive detection
The Priciple of Western Blot
-
Page 7
1.Tissue preparation
-
SDS (sodium dodecyl sulfate十二烷基硫酸钠) is a detergent (清洁 剂)(soap) that can dissolve hydrophobic（ 难溶于水）molecules but also has a negative charge attached to it.
Towbin H, et al (1979). "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.". Proc Natl Acad Sci U S A. 76 (9): 4350–4354
Page 15
4. Fluorescent detection
results
Optimized antibody concentration, lower background
Primary Ab Secondary Ab
Page 16
1:1000 1:5000
1:1000 1:20000
the binding sites
Page 19
Medical Diagnosis
• • • HIV Lyme BSE ‘ mad cow disease’
The confirmatory HIV test
HIV has been found by ELISA To confirm the result
After ‘window period’
Page 21
Western Blot for Lyme
B. burgdorferi伯疏氏螺旋体（细胞外寄生菌） confirm the diagnosis
The Western blot IgM has a specificity of 94–96% for patients with clinical symptoms of early Lyme disease
Western Blot for Research Use
Identify the nature of the protein quantitative analysis of the micromolecule antigen Epitope ห้องสมุดไป่ตู้apping Identify the process of
Page 3
History/Background Northern Blot
Developed in 1977 by George Stark and his colleagues at Stanford University Study gene expression by detection of RNA (or isolated mRNA) in a sample
Page 8
Tissue preparation
Page 9
2.Gel electrophoresis
-
SDS-PAGE(SDS变性不连续聚丙烯酰胺凝胶电泳)
Page 10
3.Transfer
-
nitrocellulose（硝酸纤维薄膜） Tips: The difference between different membranes
Page 22
Bovine spongiform encephalopathy
mad cow disease
Piron
identified and differentiated the specific molecular weights
Page 23
Page 4
History/Background Western Blot (Immunoblotting)
George Stark developed an early protein blotting technique Harry Towbin`s faster and simpler approach is clearly preferred today Electrophoretic transfer of proteins from gels to membranes
1:1000 1:100000
The applications of Western Blot
— —3127119027 马欣俐
Scientific Research
• the protein is successfully expressed ？ • the target protein is present in a given cell? • the quantity of the protein?
Southern blot (DNA)
History/Background Southern Blot
‘Southern’ hybridization named after Sir Edwin Southern Developed in 1975 One of the most highly cited scientific publications Allow the detection of a specific DNA sequence (gene or other) in a large, complex sample of DNA (e.g. cellular DNA)
The History of Western Blotting
History/Background
Spawned naming of related techniques:
Northern blot (RNA)
Western blot (Protein)
Eastern blot
Page 2
Page 11
Tissue preparation
Page 12
4.Blocking
-
Tips: one step and two steps
Page 13
Tissue preparation
one step
two steps
Page 14
5.Analysis
-
1. Colorimetric detection 2. Chemiluminescent detection 3. Radioactive detection