Methods in Signal Transduction

An excellent signaling pathway methods and techniques lab course. (Prof. Rony Seger, Department of Biological Regulation at Weizmann institute)

Content:

1) Introduction.................................................................................................................4
2) Transient Transfections................................................................................................6
2A. DNA Transfection Using DE-Dextran..........................................................6
2B. DNA Transfection Using CaPO4..................................................................6
2C. DNA Transfection using PEI……………………………………………….7
2D. DNA Transfection Using Electroporation.....................................................7
2E. DNA Transfection Using Lipofectamine.......................................................7
2F.
b
-Galactosidase Staining Protocol..................................................................8
3) General Techniques.....................................................................................................8
3A. SDS-PAGE and Western Blotting................................................................8
SDS-polyacrylamide gel electrophoresis (SDS- PAGE)..........................8
Western Blotting (protein blotting)..........................................................9
3B. Preparation of Cell Extracts...........................................................................11
Preparation of cell extracts with a detergent..............................................11
Preparation of cell extracts using sonication.............................................12
Determination of protein concentration....................................................13
4) Growth Factor Stimulated Tyr-Phosphorylation..........................................................14
4A. Introduction...................................................................................................14
4B. Use of Antibodies..........................................................................................14
4C. In vitro Tyr-kinase Assay of EGF-Receptor..................................................14
Introduction..............................................................................................14
Protein kinase assay.................................................................................15
5) Ras Assay....................................................................................................................15
5A. Introduction...................................................................................................15
5B. Preparation of Fusion Proteins......................................................................16
5C. GTPase-Fish Assay.......................................................................................17
6) IP Assay of Protein Kinases........................................................................................18
6A. Introduction (PKB/c-Akt)..............................................................................18
6B. In vitro PKB Activity Assay..........................................................................18
Immunoprecipitation................................................................................19
Phosphorylation reaction..........................................................................20
7) In-gel Kinase Assay.....................................................................................................21
8) Mini-Columns Assays.................................................................................................22
Mini-columns...........................................................................................23
Determination of kinase activity...............................................................23
9) Protein Kinase C (PKC) Activity.................................................................................23
9A. Preparation of Samples..................................................................................23
9B. PKC Activity Assay (Histone Phosphorylation)............................................24
10) JUN Kinase Assay...................................................................................................25
11) References.................................................................................................................2

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Last update 11-Sep-2006, Rating n/a of 0 votes.