Western 杂交（主要内容如下）

 

Preparing of Protein Lysates Western Blotting Far Western Blotting Semi Dry Blotting Stripping Membranes Trouble Shooting and Others

Preparing Protein Lysates

Preparing Protein Lysates for Western Blotting (Antisense Research Group)
   Yeast Lysates for Westerns (PMCI)
   Extracts for Yeast Westerns  (Herskowitz Lab
    Preparation of Brain Membrane Fractions for Western Blot (BD PharMingen)

Western Blotting

SDS/PAGE Protein Transfer (Gimila Lab)
    General Western Blot Protocol (KPL)
This procedure was written as a general protocol for use with standard Tris-Glycine SDS-PAGE gels and nitrocellulose membranes. It should give acceptable results in most situations. Optimization for particular situations may require modifying the conditions described.

・         Southerns, Westerns, and Northerns: Molecular Cloning Techniques (Brian White, MIT)
Basic theory, definition and procedures with illustration

・         Western Blotting (Howell Lab)
Procedures for preparation of cell lysate, gel, electrophoresis, transfer, antibody detection and stripping membranes.

・         Western Blots (William H. Heidcamp)

Western Blot Immunostaining (R D)
Provides very nice and general protocol including Western Blot Procedure, immunostaining Procedure (standard and rapid), helpful hints and trouble shooting guide
 

・         Western Blotting Protocol (The Cell Biology and Cytoskeleton Group, HMS)

・         Protein Gel and Staining (Gottschling Lab)

・         Western Blotting (Corbett Lab) 

・         ECM Protocols Western Blot (LTI)
General protocol for ECM assay. A mixture of protein is separated electrophoretically by SDS-PAGE, and the individual protein bands are transferred to nitrocellulose paper. Specific antibody is then used to probe for any of the bands it might bind to, and the nonbound antibody is washed away.  The bound antibody is then detected by the addition of a second antibody against the immunoglobulin species contained in the first antibody.  This second antibody is labeled with an enzyme, and the specific protein band can then be visualized by the addition of an enzyme substrate containing a color developer.

Western Blotting Using Chemiluminscence (Peter J. Hansen Lab)
   Western Blotting Analysis (BD PharMingen)
    Western Blotting with Horseradish Peroxidase Conjugates (Transduction Laboratories)
Detailed protocol of Western Blotting, from protein isolation, quantification, electrophoresis, transfer and more...
   Western Blotting with Alkaline Phosphatase Conjugates (Transduction Laboratories)
Detailed protocol of Western Blotting, from protein isolation, quantification, electrophoresis, transfer and more...
    Western Blotting with Monoclonal Antibodies (Transduction Laboratories)
Detailed protocol of Western Blotting, from protein isolation, quantification, electrophoresis, transfer and more...
    Western Blotting with Rabbit Polyclonal Antibodies (Transduction Laboratories)
Detailed protocol of Western Blotting, from protein isolation, quantification, electrophoresis, transfer and more...
    Western Blotting with Biotinylated Antibodies (Transduction Laboratories)
Detailed protocol of Western Blotting, from protein isolation, quantification, electrophoresis, transfer and more...
  

・         Alkaline Phosphatase (AP) Staining of Western Blots. (Molecular Genetics RecLab)

MAP Kinase Gel Shift Assay (PMCI)
  Anti-Phosphotyrosine Western Blotting (PMCI)
  

・         Enzyme-Assisted Immunoelectroblotitng (IEB or Western Blotting) (Molecular Biology Techniques Manual)
This protocol describes the use of horizontal blotting of simple SDS-PA gels, and subsequent detection of proteins using rabbit antisera and alkaline phosphatase-conjugated goat-anti-rabbit IgG, detected using bromo-chloro-indolyl phosphate (BCIP) and Nitro-blue tetrazolium (NBT) salts.
  

・         Protein Dot-/Slot-Blotting (Schleicher Schuell)
Dot-blots of protein simplify quantitative assays by direct application of the sample to a membrane or filter sheet without prior fractionation. The technique is used for carrying out binding assays on multiple samples, and is valuable for studies in which purification or other processing of the molecule may alter biological activity. The latter advantage makes the dot-blot technique ideal for preliminary screening to determine the effect of blotting or assay conditions on the activity of the sample.

・         Alkaline phosphatase (AP) staining of Western blots (Molecular Genetics Network Lab)

・         CDP-Star Western Blot Detection (Gimila Lab) 

Far Western Blotting

・         Far Western Blotting (Krause Lab)

Semi Dry Blotting

・         Western Blotting Using the SemiPhor Semi-Dry Transfer Unit (Goldberg Lab)
Very nice and detailed protocol with illustration

・         Semi Dry Blotting  

・         Western Transfer Using a Semidry Apparatus (PMCI)

Stripping Western Membranes

・         Stripping and Reprobing a Western Blot (KPL)

・         Stripping Western Membranes (Paul Brennan)
Reuse blot by stripping antibodies

・         Stripping Western Membranes (Monica Colitti)
Simple protocol for reusing blot

Troubleshooting and others

・         Troubleshooting Western And Dot-Blot Assays (Schleicher Schuell)

・         Tips and hints for the storage of antibodies (Synaptic Systems)

・         Too Many Bands on Western Blot (Synaptic Systems)
It is not uncommon that, contrary to the theoretical predictions, several bands are detected. Although it is possible that the antibody is not entirely specific for the protein, other factors may be responsible

Western Trouble Shooting (R D)
Offers general guide for Western blotting

 

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