Loading in 2 Seconds...
Loading in 2 Seconds...
The isolation of proteins Cell LysisFreeze/thaw and homogenization. Special detergent-based reagents Affinity PurificationCentrifugation ofcrude cell lysate. Affinity chromatography. Magnetic particles. Sample PreparationPurity 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 orderof 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.
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:Theacrylamide and bis-acrylamide are neurotoxic!!!
Before SDS Chargedparts Hidrophobeparts After SDS The effect of SDS pretreatment
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 • 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). • To remove unbound antibody, wash the membrane three times with TBST for 5 ~ 10 minutes each. • Secondary Antibody Binding • Incubate blot with Blocking Buffer /Antibody Diluent containing the appropriate antibody dilution (e.g.goat anti-human IgG-HRP conjugate) for 30 minutes. • 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
ECL detection ECL: Enhanced Chemiluminescence
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 markerLane 2 - positive patient sampleLane 3 - positive patient sampleLane 4 - monoclonal antibodies for 39 and 41kD bandsLane 5 - monoclonal antibodies for 41kD bandLane 6 - monoclonal antibodies for 39 and 41kD bandsLane 7 - monoclonal antibodies for 31 and 34kD bandsLane 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 asolid surface applied with the help of a robot in great density on activatedglasscarrier. The control of chips is performed with widely used method checking the interaction ligand-protein pair, determining level of aspecificbinding and the background. • Application for research and diagnostic (eg. monitoring the alteration in protein pattern of tumorous patient).
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.
Which of these stepsdo 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