Western blot, Western blot, Protein electrophoresis, ELISA, Protein electrophoresis, ELISA,

Protein chipProtein chip

From DNA to proteinFrom DNA to protein

Why it is easy to separate DNA?Why it is easy to separate DNA?

The structure of proteinsThe structure of proteins

The The classesclasses

of amino-of amino-acidsacids

3D structure of protein3D structure of protein

The isolation of proteinsThe isolation of proteins

Cell LysisFreeze/thaw and homogenization. Special detergent-based reagents

Affinity PurificationCentrifugation of crude 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 - antibodyAntigen - 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 Methods based on specific antigen and antibody binding antibody binding

Direct method We label the antigen or the antibody

Indirect methodWe 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” methodIn 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-blotThe essence of Western-blot

Acrylamide gelelectrophoresisAcrylamide gelelectrophoresisPretreatment 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 electrophoresisDenaturation gel with SDSTwo layers: a concentrating and a separation gel layers

ElectroblotVertical gel

Checking the efficiency of blottingCoomassie-brilliant-blue dye (CBB)

Acrylamide polymerizationAcrylamide polymerization

Caution: The acrylamide and bis-acrylamide are neurotoxic!!!

The effect of SDS pretreatmentThe effect of SDS pretreatment

Before SDS

After SDS

Charged parts

Hidrophobe parts

Vertical gel set upVertical gel set up

Running a protein separation gelRunning a protein separation gel

Staining the protein gelStaining the protein gel

STANDARD PROTOCOLSTANDARD 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 Binding1.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 Binding1.Incubate blot with Blocking Buffer /Antibody Diluent containing the appropriate antibody dilution (e.g.goat anti-human 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

BlottingBlotting

Checking the efficiency of blotting on membrane: Ponceau staining

The way from gel separation to detection The way from gel separation to detection of the wanted proteinof the wanted protein

ECL detection ECL detection

ECL: Enhanced Chemiluminescence

Alkaline phosphatase detectionAlkaline phosphatase detection

Application of Western blot for Lyme Application of Western blot for Lyme disease detectiondisease detection

Detection of bacterial proteins of Borrelia burgdorferi spreaded by tick byte in a patient blood sample

Lyme disease reactive Western blotLyme disease reactive Western blotDescription of lanes:Description of lanes:Lane 1 - molecular weight Lane 1 - molecular weight markermarkerLane 2 - positive patient Lane 2 - positive patient samplesampleLane 3 - positive patient Lane 3 - positive patient samplesampleLane 4 - monoclonal Lane 4 - monoclonal antibodies for 39 and antibodies for 39 and 41kD bands41kD bandsLane 5 - monoclonal Lane 5 - monoclonal antibodies for 41kD bandantibodies for 41kD bandLane 6 - monoclonal Lane 6 - monoclonal antibodies for 39 and antibodies for 39 and 41kD bands41kD bandsLane 7 - monoclonal Lane 7 - monoclonal antibodies for 31 and antibodies for 31 and 34kD bands34kD bandsLane 8 - positive control Lane 8 - positive control poolpool

Immunoblotting (western blotting) detects Immunoblotting (western blotting) detects proteins that have been size-fractionated on proteins that have been size-fractionated on

an electrophoresis gelan electrophoresis gel

Protein chipProtein chip

Ligand-chipLigand-chip: It is possible to detect protein pattern of a cell : It is possible to detect protein pattern of a cell by using a method based specific antigen antibody binding by using a method based specific antigen antibody binding (at DNS chips the hybridization is the basic technique)(at DNS chips the hybridization is the basic technique)

More hundred antibodies that can be immobilized on aMore hundred antibodies that can be immobilized on a solid solid surface applied with the help of asurface applied with the help of a robot robot in great density on in great density on aacctivtivatedated glassglass carriercarrier. . The control of The control of chipchipss is performed is performed with widely used method checking the interaction with widely used method checking the interaction ligand-ligand-proteinprotein p pair, determining level of aspecificair, determining level of aspecific binding and the binding and the backgroundbackground..Application for research and diagnostic (eg. monitoring the Application for research and diagnostic (eg. monitoring the alteration in protein pattern of tumorous patient).alteration in protein pattern of tumorous patient).

Detection on protein chipDetection on protein chip

Yeast protein pattern on protein chipYeast protein pattern on protein chip

AutomatizationAutomatization

ELISAELISA

The The ELISAELISA (Enzyme Linked Immunosorbent Assay) is an immunotest (Enzyme Linked Immunosorbent Assay) is an immunotest with high sensitivity, in which the antigen or the antibody is linked to a 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 solid (plastic) surface. The test generally performed in plastic plates with 96 wells (in 100-200 96 wells (in 100-200 l volume). Mostly, the the antigen is pre-absorbed l volume). Mostly, the the antigen is pre-absorbed to the plastic surface then different dilutions of the tested serum sample to the plastic surface then different dilutions of the tested serum sample (from a patient) are added to the wells.(from a patient) are added to the wells.

ApplicationApplication: : The application scale of ELISA is broad (e.g. Identification The application scale of ELISA is broad (e.g. Identification of viral and bacterial infections; the identification and quantification of of viral and bacterial infections; the identification and quantification of hormones or cytokines in blood circulation, etc). The antibodies produced hormones or cytokines in blood circulation, etc). The antibodies produced against pathogenic microorganism can be identified in blood. An infection against pathogenic microorganism can be identified in blood. An infection state can be deducted from the increase or decrease of specific antibodies. state can be deducted from the increase or decrease of specific antibodies. The test sensitivity is high, ng amount can be measured.The test sensitivity is high, ng amount can be measured.

ELISA data from three patients

                                                                        

                                                                                    

                        

Positive Control

Negative Control Patient A

Patient B

Patient C

Assay Control

1.689 0.153 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) Denaturationb) Blockingc) Hybridizationd) Antibody bindinge) Development

3. Which statement is not valid for antibodies?a) They belong to proteinsb) They can bind only specificallyc) Can be found in mother milk, too d) they have two antigen binding sites at least e) Generally can be isolated without previous immunization

5. Which component is not needed for a Western-blot?a) SDSb) mercaptoethanolc) probed) nitrocellulose filtere) agarose gel