Protein Complexes
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Tecniche immunochimiche ✓Principi generali: immunità umorale ✓Immunoglobuline: struttura, produzione anticorpi policlonali, monoclonali ✓Applicazioni: ▪ Immunoprecipitazione, immunodifusione, immunoelettroforesi, Co-IP, test di agglutinazione ▪ Dosaggi RIA, ELISA ▪ Immunoblotting: Western Blot ▪ Immunoistochimica (IHC) Microscopia fluorescenza, elettronica ▪ Citofluorimetria a flusso ▪ Impiego in terapia Determination of Ab affinity (Ka) by equilibrium dialysis. Semipermeable membrane A radioactively labeled ligand that is small to pass through (haptens, oligopeptides ) Plot of concentration of ligand in each compartment with time The difference in the two compartments Sensitivity of various immunoassays * * * * * * * * ** * * Determinanti antigenici/epitopi Complessi antigene- anticorpo Precipitation reactions in fluids yield a precipitiation curve ❖ Sensitivity ❖ Specificity Effect of Ab/Ag ratios on the formation of Ab-Ag precipitates Immunoprecipitation Immunoprecipitation (IP) is a technique to isolate a specific protein out of a solution using an antibody that binds to it. Antibody-protein complexes are removed from the solution with the addition of an insoluble form of an antibody binding protein, such as Protein A or Protein G Immunoprecipitation* *antibody conjugated to agarose or Magnetic Beads. Immunoprecipitation Immunoprecipitation assays detect the interaction of a target protein with other proteins or nucleic acids •Co-Immunoprecipitation (Co-IP) is a powerful method that is most widely used by researchers to analyze protein–protein interactions. This process provides a rapid and simple method to separate a specific protein from a sample containing thousands of different proteins, such as serum, cell lysate, homogenized tissue or conditioned media. Principle and method of co-immunoprecipitation (Co-IP) In Co-IP, complexes of two (or more) proteins are isolated using a procedure similar to the IP procedure. Co-IP is often used for the analysis of interactions of multiple proteins and their functions. The principle of Co-IP is the same as IP, except that associated proteins are precipitated. Immunoprecipitation Immunoprecipitation assays detect the interaction of a target protein with other proteins or nucleic acids. Assay examples include Co-IP, ChIP, RIP. •Chromatin Immunoprecipitation (ChIP) is a type of immunoprecipitation used to investigate regions of genome associated with a target DNA-binding protein, or conversely to identify specific proteins associated with a particular region of the genome. •RNA Immunoprecipitation (RIP) is performed with an antibody that targets a specific RNA-binding protein. By isolating the protein, the RNA bound to it is also isolated. The RNA-protein complexes are separated by RNA extraction. The RNA can be analyzed by cDNA sequencing or RT-PCR. ChIP assays Protein and associated chromatin in living cells or tissues are cross-linked using formaldehyde. The cross-linked DNA–protein complexes (chromatin- protein) are then sheared into ∼500 bp DNA fragments using either enzymatic digestion or physical shearing by sonication. The DNA-protein complexes are then immunoprecipitated by an appropriate protein-specific antibody. After the cross-links are reversed, the associated DNA fragments are eluted, which is followed by immunoprecipitation of the cross-linked complexes and analysis of the resultant DNA by endpoint or quantitative polymerase chain reaction (qPCR), or next-generation sequencing (ChIP-seq). ChIP assays can be used for the following studies: ❖DNA sequences occupied by multiple specific protein targets ❖Binding sites and distribution of a particular protein, such as a transcription factors, transcription co- factors, DNA replication factors and DNA repair proteins throughout the entire genome, under specified cellular conditions ❖Gene transcription and polymerase activity ❖Complex DNA/protein interactions underlying disease phenotypes ❖Modifications to proteins, such as histones, that may influence chromatin structure and gene expression ❖Nucleosome architecture and regulation of chromosomal maintenance Immunodiffusione in gel Diffusione radiale semplice di Mancini Radial immunodiffusion in the Ab-containing semisolid medium -> The area is proportional to the conc. of Ag The region of equivalence Diagrammatic representation of radial & double immunodiffusion. precipitation reactions in gels yield visible precipitin lines; no visible precipitate forms in regions of Ab or Ag excess. Immunodiffusione Doppia Immunoelettroforesi HS = proteine del siero umano HSA = albumina serica umana Immunoelectrophoresis an antigen mixture is first electrophoresed to separate its components by charge - diffusion & producing lines of precipitation. Immunoelectrophoresis Immunoelettroforesi bidimensionale Risultato dell’immunoelettroforesi bidimensionale di siero umano (HS): (a) con agar contenente anticorpi anti-HS nella seconda dimensione; (b) con agar contenente anticorpi anti IGM umana nella seconda dimensione Rocket Immunoelettroforesi Visualizzazione bande di precipitazione Test di fissazione del complemento Rivela la capacità di un siero di attivare la cascata del complemento in presenza di un antigene specifico per l’eventuale anticorpo del paziente Test di Agglutinazione su particelle di Lattice Anticorpi specifici verso un particolare antigene microbico vengono adsorbiti in fase solida (su particelle di lattice). Se l’antigene è presente nel campione, questo si lega all’anticorpo determinandola formazione di un agglutinato. Reazione di agglutinazione positiva. Sferette di polistirene del diametro di 1 micron rivestite di anticorpi monoclonali su un comune supporto bianco per agglutinazione poste a contatto con campioni contaminati da micoplasmi. Mediante la reazione antigene- anticorpo, in pochi minuti attorno alle sferette si generano agglomerati di micoplasmi visibili a occhio nudo. Agglutination Reactions -The original pregnancy test kit employed hapten inhibition (agglutina tion inhibition) to determine the presence or absence of human chorionic gonadotropin (HCG) -The kits currently on the market use ELISA-based assays. -Also used to determine the use of illegal drugs, & immunity (Ab) to virus (rubella). Agglutination Reactions -visible clumping by interaction between Ab & a particulate antigen such as RBC, latex beads. -depend on the crosslinking of polyvalent antigens, similar to precipitation reactions (lgM is a good agglutinin) -provide a way to type bacteria with a panel of typing antisera. -routinely performed to type RBCs for blood transfusion. + + + (control) Demonstration of humaglutination using Ab against sheep red blood cells (SRBCs): a constant # of SRBCs plus serial two-fold dilutions of anti-SRBC serum If sufficient antibody is present to agglutinate and form cross-linking with the antigen, the antibody-antigen complex forms a mat at the bottom of the well. If insufficient antibody is present, the cells roll down the sloping sides of the well to form a red pellet or "button" at the bottom of the well. Western blot Trasferimento su membrana* electroblotting *PVDF nitrocellulosa Trasferimento su membrana: esempio di Western blot Prima del trasferimento Dopo il trasferimento Gel E Membrana Il trasferimento delle proteine su di una membrana viene fatto avvenire mediante applicazione di una differenza di potenziale. Le proteine migrano in una direzione che dipende dalla loro carica. Nel caso del trasferimento di proteine separate mediante SDS-PAGE la loro migrazione è anodica dal momento che recano una carica netta negativa data dall’SDS. Perché è utile avere delle proteine immobilizzate su di una membrana? Proteine immobilizzate sulla SUPERFICIE di un supporto solido => DISPONIBILI per una serie di operazioni • Riconoscimento anticorpale • Interazioni proteina/proteina Western blot Workflow Semidry Nitrocellulosa 0.22 mm PAGE Colorazione Saturazione Utilizzo Wet PVDF 0.45 mm Western blotting (blotting di proteine) Rivelazione enzimatica di antigeni separati elettroforeticamente Anche : Comunemente: Anticorpi secondari marcati con 125I, con Anticorpi secondari coniugati ad un enzima fluoresceina, oro, biotinilati. Gli enzimi più utilizzati (con prodotti precibitabili) : AP (Alkaline Phosphatase) HRP (Horse Radish Peroxidase) BCIP/NBT 4-cloro-1-naftolo, 5-Bromo-4-Chloro-3’-Indolyphosphate p- Toluidine Salt / Nitro-Blue Tetrazolium Chloride oppure luminolo Enhanced Chemi Luminescence (ECL) SDS PAGE and Western Blot to detect a protein of interest Enhanced Chemi Luminescence (ECL) Effect of oxidative agents on the Prx expression in S. solfataricus Western blot H2O2 Paraquat tert-butyl hydroperoxide 0 30 60 120 0 30 60 120 0 30 60 120 Prx The peroxiredoxin is up regulated by the different stressors Ascaris suum cytochrome b5, an adult-specific secretory protein reducing oxygen-avid ferric hemoglobin Hashimoto M. et al. Archives of Biochemistry and Biophysics 471 (2008)) 42–49 Western blot ➢ Nell’ adulto di Ascaris suum il citocromo b5 è espresso solo nella forma matura e solo nella frazione citosolica e nel fluido perienterico ➢ Contrariamente a quanto precedentemente ipotizzato è assente nei mitocondri e nei microsomi Una applicazione clinica del Western Blot Viral Proteins HIV, like any other virus, is composed of a number of different proteins. The Western Blot positive control lane contains proteins from patient sera as well as HIV proteins. HIV positivity can therefore only be confirmed by the presence of the following types of proteins: gp160 viral envelope