蛋白质电泳-实验方法

蛋白质电泳(主要内容如下)

  • One-Dimensional SDS-PAGE

  • Two-Demensional SDS-PAGE

  • Protein Electrophoresis in Agarose Gel 

  • Gel Staining

  • Recipes

One-Dimensional SDS-PAGE

      Protein Gel and Staining (Gottschling Lab)
Provides procedures for gel preparation, gel staining...

     Preparation of SDS-Polyacrylamide Gels (SDS-PAGE) (William H. Heidcamp)

     SDS Gel Electrophoresis (Petra Klaff)
Resolving and stacking gel

     SDS-Page Gel Electrophoresis (Dr. Chastain)

     SDS PAGE Gels (Gimila Lab)

     SDS-Polyacrylamide gels (NWFSC)
Standard Laemmli protocol

     Denaturing Discontinuous Polyacrylamide Gel Electrophoresis (SDS-PAGE) (Goldberg Lab)
Very nice and detailed protocol

  • PolyAcrylamide Gel Electrophoresis (PAGE) gels
    Preparation of stock solutions of acrylamide and method of preparation of convex,  and gradient gels
      

     SDS-PAGE Gels (PMCI Research)
Detailed protocol for preparing gel, reagents for SDS-PAGE and mini-gels

        Tricine-Polyacrylamide Gels (NWFSC)
For high resolution of small proteins

        Tricine SDS-PAGE (Goldberg Lab)

Detailed protocol  

        Electroblotting of SDS-PAA gels (Molecular Genetics Network Lab)

  • SDS-polyacrylamide gels (single or double Minigel system) (Molecular Genetics Network Lab)
        Electroelution of Proteins From SDS-PAGE Gels (Mike A. Dyer)

This is a general protocol that was developed for the ISCO electroeluter but could easily be applied to other systems.

Two-Demensional SDS-PAGE

        2-D PAGE Protein Analysis (ExPASy)

        Analytical 2-D PAGE protocols

        Preparative 2-D PAGE protocols

       Post-separation analysis  

        Isoelectric Focusing as the First Dimension (UCSF Tumor Immunology)

        SDS-PAGE as the Second Dimension (UCSF Tumor Immunology)
In this method, proteins are highly denatured and associated with the anionic detergent SDS. Proteins run through the stacking gel, then stack at the membrane where the ion fronts compact them. Upon entering the separating gel, the proteins become separated with lower MW proteins running faster than higher MW proteins.
  

         Analysis of Proteins using Small Format 2D Gel Electrophoresis (Dr Phillip Cash)
Detailed protocol on 2D protein electrophoresis. 

Protein Electrophoresis in Agarose Gel 

         Protein Electrophoresis in Agarose Gels (FMC)
In certain circumstances, electrophoresis of proteins in agarose gels has distinct advantages when compared to polyacrylamide gels. This protocol describes procedures for gel casting, sample preparation, and protein staining and recovery.

Gel Staining

        SDS PAGE Staining Protocol (LTI)
General method for silver stain and coomassie blue stain with recipes

        Coomasie Blue Staining of Protein Gels (William H. Heidcamp)
Coomasie Brilliant Blue R 250 is the most commonly used staining procedure for the detection of proteins. It is the method of choice if SDS is used in the electrophoresis of proteins, and is sensitive for a range of 0.5 to 20 micrograms of protein.

        Silver Staining of Protein Gels (William H. Heidcamp)

        Staining of Polyacrylamide Gels (UCSF Tumor Immunology)

       Coomassie G250 

       Fast Stain (Zoion) 

       Imidazole-Zinc
  

        Silver staining of PAGE gels

        Silver Staining of Acrylamide Gels (Waters Lab)

        Silver Stain for SDS PAGE (Hahn Lab)

        Silver staining of protein gels

        Cupric Chloride Staining : for SDS-PAGE gels (Mike A Dyer)
This protocol is 2-3 times more sensitive than coomasie blue staining, it is much quicker, and the gels can be stored at 4° for many months without protein degredation.

        Ethanol-based SDS-PAGE Coomassie Blue staining (NWFSC)

        Direct Densitometry of Protein Gels (William H. Heidcamp)

Recipes

        SDS-PAGE Recipes (The Cell Biology and Cytoskeleton Group, HMS)