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Transcript
Western blot, Protein electrophoresis, ELISA, Protein chip From DNA to protein Why it is easy to separate DNA? The structure of proteins The classes of aminoacids 3D structure of protein The isolation of proteins Cell Lysis Freeze/thaw and homogenization. Special detergent-based reagents Affinity Purification Centrifugation of crude cell lysate. Affinity chromatography. Magnetic particles. Sample Preparation Purity and concentration checkup The methods described apply to preparation of samples for use in many common laboratory techniques such as electrophoresis, Western blotting, ELISA, mass spectrometry, enzyme activity assays and more. Antigen - antibody Antigens: objects, recognized as foreign by vertebral organisms and can challenge the immune system to produce specific antibodies and immune cells against antigens. The order of immuno-stimulant strength: proteins >polysaccharides > lipids > nucleicacids. Antibodies: Immunoglobulins produced by the B cells of the immune system. They recognize and react with the antibodies specifically. The antibodies can be separated from the gammaglobulin fraction of the blood serum. Methods based on specific antigen and antibody binding Direct method We label the antigen or the antibody Indirect method We detect the antigen-antibody binding with a labelled anti-immunglobulin antibody (e.g. goat anti-human IgG) that recognize the specifically reacting primary antibody mutatjuk ki. Method is mainly applied to detect antigen specific antibodies and for their. Increased specificity. Double antibody: „sandwich” method In this method we bind the antibody-molecules reacting specifically with the antigen to solid phase. The anchored antibody specifically binds the antigen, thus the antigen isolated from multicomponent solution. The antibody-antigen binding is the detected by another specifically reacting labelled antibody. The essence of Western-blot Acrylamide gelelectrophoresis Pretreatment of protein sample for running (denaturation) Before the electrophoresis add detergent (SDS, sodium-dodecilsulfate) and disulfide bridges reducingagent (mercaptoethanol) to samples and apply heat treatment (3-5 min., 90°C). Polyacrilamide electrophoresis Denaturation gel with SDS Two layers: a concentrating and a separation gel layers Electroblot Vertical gel Checking the efficiency of blotting Coomassie-brilliant-blue dye (CBB) Acrylamide polymerization Caution: The acrylamide and bis-acrylamide are neurotoxic!!! The effect of SDS pretreatment Before SDS Charged parts Hidrophobe parts After SDS Vertical gel set up Running a protein separation gel Staining the protein gel STANDARD PROTOCOL Blocking of membrane ( blot ) To saturate nonspecific protein binding sites, incubate the nitrocellulose and PVDF membranes membrane for 30-60 minutes in Blocking Buffer ( TBST containing 1% BSA or 5 % skimmed milk). Primary Antibody Binding 1.To add primary antibody, replace the blocking solution ( which can be re-used several times ) with Blocking Buffer containing appropriate dilution of primary antibody. Incubate the blot for 30 ~ 60 minutes with gentle agitation at room temperature (or overnight at 2~ 8 °C). 2.To remove unbound antibody, wash the membrane three times with TBST for 5 ~ 10 minutes each. Secondary Antibody Binding 1.Incubate blot with Blocking Buffer /Antibody Diluent containing the appropriate antibody dilution (e.g.goat antihuman IgG-HRP conjugate) for 30 minutes. 2.Wash the blot with TBST three times for 10 minutes each to remove unbound secondary antibody. Development of signal Add alkaline phosphatase subtrate, HRP substrate, ECL substrate Blotting Checking the efficiency of blotting on membrane: Ponceau staining The way from gel separation to detection of the wanted protein ECL detection ECL: Enhanced Chemiluminescence Alkaline phosphatase detection Application of Western blot for Lyme disease detection Detection of bacterial proteins of Borrelia burgdorferi spreaded by tick byte in a patient blood sample Lyme disease reactive Western blot Description of lanes: Lane 1 - molecular weight marker Lane 2 - positive patient sample Lane 3 - positive patient sample Lane 4 - monoclonal antibodies for 39 and 41kD bands Lane 5 - monoclonal antibodies for 41kD band Lane 6 - monoclonal antibodies for 39 and 41kD bands Lane 7 - monoclonal antibodies for 31 and 34kD bands Lane 8 - positive control pool Immunoblotting (western blotting) detects proteins that have been size-fractionated on an electrophoresis gel Protein chip Ligand-chip: It is possible to detect protein pattern of a cell by using a method based specific antigen antibody binding (at DNS chips the hybridization is the basic technique) More hundred antibodies that can be immobilized on a solid surface applied with the help of a robot in great density on activated glass carrier. The control of chips is performed with widely used method checking the interaction ligandprotein pair, determining level of aspecific binding and the background. Application for research and diagnostic (eg. monitoring the alteration in protein pattern of tumorous patient). Detection on protein chip Yeast protein pattern on protein chip Automatization ELISA The ELISA (Enzyme Linked Immunosorbent Assay) is an immunotest with high sensitivity, in which the antigen or the antibody is linked to a solid (plastic) surface. The test generally performed in plastic plates with 96 wells (in 100-200 l volume). Mostly, the the antigen is pre-absorbed to the plastic surface then different dilutions of the tested serum sample (from a patient) are added to the wells. Application: The application scale of ELISA is broad (e.g. Identification of viral and bacterial infections; the identification and quantification of hormones or cytokines in blood circulation, etc). The antibodies produced against pathogenic microorganism can be identified in blood. An infection state can be deducted from the increase or decrease of specific antibodies. The test sensitivity is high, ng amount can be measured. ELISA data from three patients Positive Control Negative Control 1.689 0.153 Patient A Patient B Patient C Assay Control O.055 0.412 1.999 0.123 Partially purified, inactivated HIV antigens pre-coated onto an ELISA plate Patient serum which contains antibodies. If the patient is HIV+, then this serum will contain antibodies to HIV, and those antibodies will bind to the HIV antigens on the plate. Anti-human immunoglobulin coupled to an enzyme. This is the second antibody, and it binds to human antibodies. Chromogen or substrate which changes color when cleaved by the enzyme attached to the second antibody. 1. Which of these steps do not belong to a Western-blot? a) Denaturation b) Blocking c) Hybridization d) Antibody binding e) Development 5. Which component is not needed for a Western-blot? a) SDS b) mercaptoethanol c) probe d) nitrocellulose filter e) agarose gel 3. Which statement is not valid for antibodies? a) They belong to proteins b) They can bind only specifically c) Can be found in mother milk, too d) they have two antigen binding sites at least e) Generally can be isolated without previous immunization