* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Western Blotting
Survey
Document related concepts
Complement system wikipedia , lookup
Adoptive cell transfer wikipedia , lookup
Adaptive immune system wikipedia , lookup
DNA vaccination wikipedia , lookup
Immunocontraception wikipedia , lookup
Anti-nuclear antibody wikipedia , lookup
Duffy antigen system wikipedia , lookup
Molecular mimicry wikipedia , lookup
Immunoprecipitation wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
Immunosuppressive drug wikipedia , lookup
Transcript
Concept of Ag-Ab immunological technique • Using the characteristic of high affinity and specificity of antibody, we can detect or quantitative the antigen. Keep in mind that the antibody is protein, can also be recognized as an antigen. The major principle to determine the antigen-antibody interaction is to separate the bound form of antigenantibody complex from the free form of either antigen or antibody. Antibodies Analytical Techniques Utilizing Antibodies: • flow cytometry • gel electrophoresis • immunoprecipitation (IP) • immunoblotting • microscopy • immunofluorescence (IFA) • electron microscopy • ELISA • antibodies bind proteins with high specificity and affinity • affinity chromatography • analytical techniques CNBr Western Blotting Western Blotting • Protein denature (SDS) • SDS-PAGE gel electrophoresis • Blotting (transfer) • Blocking (BSA) • Staining with Coomasie or Ponceau S (checking) • 1° Ab serum (probe) • Washes • 2 ° Ab serum • Washes • Color development Why do proteins stick to the membrane? Hydrophobic & charge interactions. Vertical Gel Electrophoresis Prep and Run Samples Example of Western Blot Result Blot interpretation 1. 2. 3. 4. 5. Lane 1, HIV+ serum (positive control) Lane 2, HIV- serum (negative control) Lane A, Patient A Lane B, Patient B Lane C, Patient C Enzyme-Mediated Detection Enzyme Horseradish Peroxidase (HRP) Substrate Abbrev. Color Diaminobenzidine DAB Brown Enzyme Alkaline Phosphatase (AP) Substrate Abbrev. Bromochloroindolylphosphate Nitro Blue Tetrazolium BCIP (AP substrate) Purple NBT (Enhance color) Color TUGAS: 1. Komposisi dan fungsi dari masingmasing bahan pada transfer buffer 2. Methode secara rinci western blotting Immunopreciptation: Identification of protein-protein interactions Steps: 1. Attach antibody to beads via protein A 2. Lyse cells to release antigen and its binding partners 3. Mix cell lysate + antibody-coated beads (antibody binds antige 4. Purify antigen and its binding partners by centrifugation bead protein A primary antibody Immunoprecipitation • affinity purification based on isolation of Ag-Ab complexes • analyze by gel electrophoresis • initially based on centrifugation of large supramolecular complexes • [high] and equal amounts • isolation of Ag-Ab complexes • fixed S. aureus • protein A-agarose • protein G-agarose Bacterial proteins that bind IgG (Fc): • protein A (Staphylococcus aureus) • protein G (Streptococcus) • binds more species and subclasses Typical IP Protocol 1. Solubilize antigen • usually non-denaturing • SDS + excess of TX100 2. Mix extract and Ab 3. Add protein G-agarose, etc 4. Extensively wash 5. Elute with sample buffer 6. SDS-PAGE 7. Detection • protein stain • radioactivity agaroseG Radiolabeling of Proteins • carried out before IP • metabolic (amino acids or other precursors + cells) • chemically (eg., iodination) • IP and SDS-PAGE • detect by autoradiography or fluorography following electrophoresis • also provides information about synthesis, posttranslational events, etc. Western Blot vs Immunoprecipitation • Experimental Design • eg., synthesis (IP) • Ag concentration • IP better for low abundance proteins • Ag solubility • Western for insoluble proteins • Ab recognition • conformational dependent epitopes • 4o structure Basics of Immunohistochemistry 03/2005 Immunofluorescence Microscopy What is Immunohistochemistry? CELLULAR ANTIGENS Sensory Metabolic Adhesion Outline of Procedure Fixwholemount, embed and section tissue (or treat as ”” preparation – small specimens only, such as cultured cells) Wash sections in physiological buffer, e.g. PBS Incubate with protein solution (BSA or normal serum) to reduce non-specific binding of antibody to specimen (”blocking”- important!) Incubate with antibody specific to antigen in question (”primary antibody”). Include positive and negative controls (!) Wash in physiological buffer Apply suitable detection system (see below) Mount specimens and analyse microscopically Fixing and Sectioning of Tissue Common fixation methods are chemical fixation (e.g. paraformaldehyde) or cryofixation (i.e. rapid freezing). Fixation serves to preserves tissue structure and properties of antigen Tissues can be embedded in wax or resins for sectioning, or cut in the frozen state in a cryostat. The method chosen for fixation and sectioning are dependent on the properties of the tissue, antigen and the antibody used in the procedure Immunohistochemical Detection Methods Through fluorescent substances (fluorophores) –”Immunofluorescence technique” Through enzymatic conversion (often by horseradish peroxidase, HRP) of a soluble substrate (chromogen) into a non-soluble and colourful reaction product - ”Immunoenzyme technique” Immunofluorescence Fluorescence or confocal microscope necessary for analysis of specimens High structural resolution possible Advanced image reconstruction (3D) and signal quantification possible Multiple labelling easy Limited shelf life of labelled specimens Method of choice for labelling of live cells Direct Immunofluorescence Method Easy application, only few steps Not very sensitive Not very versatile, as primary antibodies need to be directly labelled with fluorophore Fluorochrome-conjugated Primary Antibody Antigen expressing Cell Indirect Immunofluorescence Method More steps involved More sensitive More versatile, as only secondary antibodies need to be labelled and different combinations of fluorophores are possible in multiple labelling experiments Fluorochrome-conjugated Secondary Antibody (anti species X Primary Antibody (from species X) Antigen expressing Cell Indirect Immunfluorescence Using Biotinylated secondary Antibody Biotin (Vitamin B) binds with high affinity to Avidin – thus good linker system Very high sensitivity Endogenous biotin may be present in tissue – risk of background Fluorochrome-conjugated Streptavidin Biotinylated Secondary Antibody (anti species X Primary Antibody (from species X) Antigen expressing Cell Multiple Immunofluorescence Multiple Labelling of a Tissue Section Live Labelling of Cultured Cells Enzymatic detection methods Brightfield microscope sufficient for analysis of specimens Suitable for tissue analysis at low magnification Resolution of subcellular structures not as good as with fluorescence methods, but can be combined with electron microscopy Unimited shelf life of labelled specimens Substrate reagents often toxic/carcinogenic Immunoezyme Labelling of Tissue Section ABC (Avidin Biotin Complex) Method Biotin (Vitamin B) binds with high affinity to Avidin – thus good linker system Extremely high sensitivity Endogenous biotin my be present in tissue – risk of background Substrate-chromogen solution Enzyme-conjugated Avidin-Biotin Complex Biotinylated Secondary Antibody (anti species X) Primary Antibody (species X) Antigen expressing Cell Common Problems Non-specific binding of primary or secondary antibodies to tissue sample; e.g. through ionic or hydrophobic interactions or binding of antibodies to free amino groups: „Background staining“ Cross-reactivity of antibodies with unrelated antigens present in tissue sample Good Resources Dako On-line Immunohistochemistry Handbook (http://www.dakousa.com/ihcbook/hbcontent.htm) NIH Immunohistochemistry Protocols (http://dir.niehs.nih.gov/dirlep/immuno.html)