Immunoprecipitation (IP) - Creative Diagnostics
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Immunoprecipitation (IP) is one of the most widely used antibody-based techniques. It is used to purify and enrich the protein of interest from a complex mixture such as cell lysate, tissue homogenate or blood sample. For more information at http://www.creative-diagnostics.com/immunoprecipitation-guide.htm
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Therapeutic drug monitoring (TDM) measures drug concentrations in blood or plasma to optimize drug dosage and ensure maximum therapeutic benefit with minimal toxic effects. TDM is indicated for drugs with a narrow therapeutic index, life-threatening diseases, or those affected by individual variability. Factors like age, organ function, drug interactions, and compliance can impact drug levels. The TDM process involves collecting samples at specific times, analyzing drug concentrations using methods like chromatography, and interpreting results with clinical context to guide prescribing. Common drugs monitored include antiepileptics, immunosuppressants, and antibiotics.
IMMUNOTECHNIQUES (immunoprecipitation)
Immunoprecipitation is a technique used to purify a specific protein from a complex mixture using an antibody. It can be used to study protein function and protein-protein complexes. There are three main types: individual protein immunoprecipitation isolates a single antigen; protein complex immunoprecipitation isolates an antigen along with any bound proteins; and pull down assays investigate protein interactions using a non-antibody affinity system. The procedure involves preparing the sample and antibody, forming an immune complex, capturing the complex, and eluting the purified protein. Immunoprecipitation has applications in studying cancer, cell signaling, diseases, and more.
Immunoprecipitation and Coimmunoprecipitation
Immunoprecipitation uses an antibody to isolate an antigen of interest from a solution. The antibody binds to the antigen, and the antibody-antigen complex is pulled out of the sample using protein A/G beads. Co-immunoprecipitation identifies interaction partners of a protein of interest by preparing an antibody against a known protein, allowing the antibody to bind the protein complex, then precipitating the complex using beads coupled to protein A/G. Mass spectroscopy can then identify the specific proteins present in the complex.
IMMUNOPRECIPITATION
This document summarizes the technique of immunoprecipitation. It describes immunoprecipitation as a method to precipitate protein antigens out of solution using an immobilized specific antibody. The document outlines different applications of immunoprecipitation including measuring protein molecular weight, detecting post-translational modifications, and analyzing protein-protein interactions. It also describes different types of immunoprecipitation techniques such as co-immunoprecipitation, chromatin immunoprecipitation, and RNA immunoprecipitation.
Immunoassays
Radioimmunoassay (RIA) and enzyme immunoassay (EIA) techniques allow for the quantitative detection of analytes at trace levels. RIA uses radioisotopes as labels, while EIA uses enzymes. Both techniques can be formatted competitively or non-competitively. Variations include immunoradiometric assays, enzyme-multiplied immunoassay technique (EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and apoenzyme reactivation immunoassay (ARIS). These assays find wide application in research, clinical medicine, and drug monitoring due to their high sensitivity and specificity.
Immunoprecipitation: Procedure, Analysis and Applications
Immunoprecipitation is a precipitaion technique which allows the isolation of protein or protein complex from biological samples.
Incubate sample with antibody against protein of interest.
Separate antibody-protein complex from remaining sample
Analysis
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ELISA - Enzyme linked immunosorbent assay
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Definition
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Therapeutic drug monitoring (TDM) measures drug concentrations in blood or plasma to optimize drug dosage and ensure maximum therapeutic benefit with minimal toxic effects. TDM is indicated for drugs with a narrow therapeutic index, life-threatening diseases, or those affected by individual variability. Factors like age, organ function, drug interactions, and compliance can impact drug levels. The TDM process involves collecting samples at specific times, analyzing drug concentrations using methods like chromatography, and interpreting results with clinical context to guide prescribing. Common drugs monitored include antiepileptics, immunosuppressants, and antibiotics.
IMMUNOTECHNIQUES (immunoprecipitation)
Immunoprecipitation is a technique used to purify a specific protein from a complex mixture using an antibody. It can be used to study protein function and protein-protein complexes. There are three main types: individual protein immunoprecipitation isolates a single antigen; protein complex immunoprecipitation isolates an antigen along with any bound proteins; and pull down assays investigate protein interactions using a non-antibody affinity system. The procedure involves preparing the sample and antibody, forming an immune complex, capturing the complex, and eluting the purified protein. Immunoprecipitation has applications in studying cancer, cell signaling, diseases, and more.
Immunoprecipitation and Coimmunoprecipitation
Immunoprecipitation uses an antibody to isolate an antigen of interest from a solution. The antibody binds to the antigen, and the antibody-antigen complex is pulled out of the sample using protein A/G beads. Co-immunoprecipitation identifies interaction partners of a protein of interest by preparing an antibody against a known protein, allowing the antibody to bind the protein complex, then precipitating the complex using beads coupled to protein A/G. Mass spectroscopy can then identify the specific proteins present in the complex.
IMMUNOPRECIPITATION
This document summarizes the technique of immunoprecipitation. It describes immunoprecipitation as a method to precipitate protein antigens out of solution using an immobilized specific antibody. The document outlines different applications of immunoprecipitation including measuring protein molecular weight, detecting post-translational modifications, and analyzing protein-protein interactions. It also describes different types of immunoprecipitation techniques such as co-immunoprecipitation, chromatin immunoprecipitation, and RNA immunoprecipitation.
Immunoassays
Radioimmunoassay (RIA) and enzyme immunoassay (EIA) techniques allow for the quantitative detection of analytes at trace levels. RIA uses radioisotopes as labels, while EIA uses enzymes. Both techniques can be formatted competitively or non-competitively. Variations include immunoradiometric assays, enzyme-multiplied immunoassay technique (EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and apoenzyme reactivation immunoassay (ARIS). These assays find wide application in research, clinical medicine, and drug monitoring due to their high sensitivity and specificity.
Immunoprecipitation: Procedure, Analysis and Applications
Immunoprecipitation is a precipitaion technique which allows the isolation of protein or protein complex from biological samples.
Incubate sample with antibody against protein of interest.
Separate antibody-protein complex from remaining sample
Analysis
ENZYME LINKED IMMUNOSORBENT IMMMUNO ASSAY
This presentation explains about the principle and procedure involved in elisa method of immunoassay, development o f elisa , application advantages and disadvantages of elisa
ELISA - Enzyme linked immunosorbent assay
Enzyme linked immunosorbent assay
History
Definition
Principle
Components
Types
Pros and cons
Troubleshooting
Applications
Modifications of ELISA
Antibody
The document discusses the principles of antibody production, including how antibodies are produced by B cells in response to antigens, the process of antibody production including antigen presentation, activation of helper T cells and B cells, proliferation of plasma cells, and the role of memory cells in the secondary response. It also covers the structure and classes of antibodies, as well as the production and uses of monoclonal and polyclonal antibodies in research, diagnosis and therapy.
Immunoprecipitation
Precipitation reactions occur when an antibody and soluble antigen interact in solution, forming a visible precipitate. A precipitin curve can be created by plotting the amount of precipitate formed against increasing concentrations of antigen. Immunoelectrophoresis techniques separate antigens by charge and detect antigen-antibody reactions using precipitation lines formed in an agar gel containing antibodies. Crossed immunoelectrophoresis further separates proteins through two perpendicular rounds of electrophoresis and antibody detection.
Immunoprecipitation
Immunoprecipitation is a technique to isolate a specific protein from a mixture using an antibody that binds to that protein. There are two main methods: traditional IP and crosslink IP. Traditional IP recovers both the antibody and antigen, which can make identifying the antigen difficult if they have similar sizes. Crosslink IP covalently attaches the antibody to beads, allowing isolation of just the antigen without antibody contamination to easily identify and analyze the target protein. However, in some rare cases the traditional method may work better, such as if the antibody is unstable or the antibody-antigen binding is too strong for elution.
Antigen antibody reactions
1) Affinity refers to the strength of binding between a single antigenic site and antibody binding site, while avidity refers to the overall strength of binding between multivalent antigens and antibodies.
2) Avidity is influenced by both affinity and the valence (number of binding sites) of the antigen and antibody. More binding sites results in higher avidity.
3) Specificity refers to an antibody only binding to one antigen, while cross-reactivity is binding to multiple antigens that share similar epitopes.
Immunoprecipitation
Immunoprecipitation is a method used to isolate a specific antigen from a mixture using the antigen-antibody interaction. It involves adding an antibody to a mixture containing the antigen and incubating to allow antigen-antibody complexes to form. These complexes are then incubated with immobilized secondary antibodies or protein A/G beads. Antigens isolated through immunoprecipitation can be analyzed by SDS-PAGE or Western blotting. It has various applications including isolating and detecting proteins of interest, studying protein-protein interactions, and identifying unknown proteins in complexes.
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Immunoprecipitation is a technique used to isolate a protein of interest from a complex protein mixture using an antibody that specifically binds to that protein. The key steps involve lysing cells, incubating the sample with the target antibody, precipitating the antibody-protein complex, washing away non-specific bindings, and then analyzing the isolated proteins. Immunoprecipitation can be used to study protein-protein interactions, identify proteins in complexes, and enrich low abundance proteins for further analysis.
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Immunoelectrophoresis
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Antibody
The document discusses the principles of antibody production, including how antibodies are produced by B cells in response to antigens, the process of antibody production including antigen presentation, activation of helper T cells and B cells, proliferation of plasma cells, and the role of memory cells in the secondary response. It also covers the structure and classes of antibodies, as well as the production and uses of monoclonal and polyclonal antibodies in research, diagnosis and therapy.
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Precipitation reactions occur when an antibody and soluble antigen interact in solution, forming a visible precipitate. A precipitin curve can be created by plotting the amount of precipitate formed against increasing concentrations of antigen. Immunoelectrophoresis techniques separate antigens by charge and detect antigen-antibody reactions using precipitation lines formed in an agar gel containing antibodies. Crossed immunoelectrophoresis further separates proteins through two perpendicular rounds of electrophoresis and antibody detection.
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Immunoprecipitation is a technique to isolate a specific protein from a mixture using an antibody that binds to that protein. There are two main methods: traditional IP and crosslink IP. Traditional IP recovers both the antibody and antigen, which can make identifying the antigen difficult if they have similar sizes. Crosslink IP covalently attaches the antibody to beads, allowing isolation of just the antigen without antibody contamination to easily identify and analyze the target protein. However, in some rare cases the traditional method may work better, such as if the antibody is unstable or the antibody-antigen binding is too strong for elution.
Antigen antibody reactions
1) Affinity refers to the strength of binding between a single antigenic site and antibody binding site, while avidity refers to the overall strength of binding between multivalent antigens and antibodies.
2) Avidity is influenced by both affinity and the valence (number of binding sites) of the antigen and antibody. More binding sites results in higher avidity.
3) Specificity refers to an antibody only binding to one antigen, while cross-reactivity is binding to multiple antigens that share similar epitopes.
Immunoprecipitation
Immunoprecipitation is a method used to isolate a specific antigen from a mixture using the antigen-antibody interaction. It involves adding an antibody to a mixture containing the antigen and incubating to allow antigen-antibody complexes to form. These complexes are then incubated with immobilized secondary antibodies or protein A/G beads. Antigens isolated through immunoprecipitation can be analyzed by SDS-PAGE or Western blotting. It has various applications including isolating and detecting proteins of interest, studying protein-protein interactions, and identifying unknown proteins in complexes.
Immunofluorescence: A Brief Discussion
Immunofluorescence is a technique that uses the binding of antibodies to antigens to detect the presence of substances. It relies on tagging antibodies with fluorescent dyes called fluorophores. When exposed to ultraviolet light, the fluorophore-tagged antibody complexes emit visible light that can be seen using a fluorescent microscope. There are several types of immunofluorescence assays including direct, indirect, and quantitative assays that are used to detect microorganisms, antibodies, and other biomolecules through the antigen-antibody reaction and fluorescent signal produced.
Bacterial vaccines
There are several types of bacterial vaccines that work through different mechanisms of the immune system. Active immunization stimulates the body's own immune response by introducing an antigen from the bacteria. This induces long-term immunity through memory B and T cells. Passive immunization provides immediate, short-term protection by transferring pre-formed antibodies. Common bacterial vaccines include those targeting Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, diphtheria, tetanus, pertussis, Lyme disease, anthrax, tuberculosis, and typhoid fever. These vaccines contain inactivated toxins, purified proteins, capsular polysaccharides, or live attenuated bacterial strains.
Immunoprecipitation
Immunoprecipitation is a technique used to isolate a protein of interest from a complex protein mixture using an antibody that specifically binds to that protein. The key steps involve lysing cells, incubating the sample with the target antibody, precipitating the antibody-protein complex, washing away non-specific bindings, and then analyzing the isolated proteins. Immunoprecipitation can be used to study protein-protein interactions, identify proteins in complexes, and enrich low abundance proteins for further analysis.
Ria
Radioimmunoassay is an immunoassay technique that uses radiolabeled antigens or antibodies to detect and quantify antigens or antibodies in a sample. It involves competitive binding between the radiolabeled and unlabeled antigens or antibodies. The amount of radiolabeled antigen or antibody bound is inversely proportional to the concentration of the unlabeled antigen or antibody in the sample. RIA is highly sensitive and specific due to the immune reaction between antigens and antibodies. It has applications in endocrinology, pharmacology, oncology, and epidemiology to detect hormones, vitamins, drugs, and infectious disease markers.
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This document describes single radial immunodiffusion (RID), a quantitative technique used to determine the concentration of an antigen in a sample. RID involves incorporating specific antibody into an agarose medium with a central well for the antigen sample. The antigen diffuses radially and reacts with the antibody, forming a visible precipitate ring whose diameter relates to the antigen concentration. The technique is simple, cost-effective, and can quantify immunoglobulins, complement components, or other antigens from biological fluids by measuring ring diameters against a standard curve.
Isotachophoresis & ief(iso electric focusing
Isotachophoresis (ITP) and isoelectric focusing (IEF) are electrophoretic techniques used to separate ionic analytes. ITP separates analytes based on differences in ionic mobility in a discontinuous electrolyte system, resulting in sharpened zones. IEF separates molecules like proteins based on differences in their isoelectric point (pI) by exploiting the fact that a molecule's overall charge is dependent on the pH of its surroundings. Both techniques are useful for applications like protein analysis, metabolite detection, and environmental analysis.
Immunoelectrophoresis
Electrophoresis is a technique used to separate charged particles like proteins and antibodies through a gel or liquid using an electric field. The document describes how electrophoresis works to separate antigen mixtures by electric charge. Antiserum is then added to wells cut into an agarose gel, allowing antibodies and antigens to diffuse towards each other. Where they meet in the proper proportions, lines of precipitation will form, indicating the presence of antigen-antibody complexes. Immunoelectrophoresis specifically uses this technique to detect the presence of antibodies and determine levels of immunoglobulins in serum to diagnose immunodeficiency diseases or identify overproduction of certain proteins.
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contains introduction on Gel eletrophoresis,principle, its types and applications of Gel electrophoresis in compact manner.
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Immunoassays powerpoint presentaion
Immunoassays are biochemical methods that use the specificity of antigen-antibody reactions to detect and quantify target molecules in biological samples. The document defines immunoassay and provides details on various types including enzyme immunoassays (ELISA, EMIT), radioimmunoassay (RIA), counting immunoassay (CIA), fluoroimmunoassay (FIA), and chemiluminescence immunoassay (CLIA). It describes the principles, components, procedures, and applications of each type of immunoassay for detecting molecules like antibodies, hormones, and proteins.
ELISA
This document discusses various types of labeled immunoassays, including fluorescent, radioactive, chemiluminescent, and enzyme labels. It describes the key characteristics and procedures for competitive and noncompetitive assays. Specific enzyme immunoassay techniques are covered in detail, such as competitive and noncompetitive ELISA, capture (sandwich) assays, rapid immunoassays, and homogeneous assays. The advantages of enzyme immunoassays include safety compared to radioimmunoassays and the ability to perform assays without expensive instrumentation. Challenges include potential interference from inhibitors or nonspecific binding.
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Western blotting is a technique used to detect specific proteins in a sample based on antibody-protein binding. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify a targeted protein. The process includes denaturing proteins, separating them by SDS-PAGE gel electrophoresis based on size, transferring to a membrane, blocking non-specific binding sites, incubating with primary and secondary antibodies, and detecting the targeted protein.
rabies vaccine and tetanus anti serum FOR PHARM ANALYSIS
- The document summarizes a seminar presentation on the biological testing and assays of rabies vaccine and tetanus anti-serum.
- It describes the Rabies Fluorescent Focus Inhibition Test (RFFIT) used to determine the rabies virus neutralizing antibody titer or potency in a vaccine. Biological assays are also used to determine the potency of tetanus anti-toxin by comparing its ability to protect mice from tetanus toxin to that of a reference standard.
- The assays involve injecting mice with serial dilutions of the vaccine or anti-serum and a fixed dose of rabies or tetanus challenge virus/toxin, and observing for symptoms to calculate
Direct ELISA Protocol
The enzyme-linked immunosorbent assay (ELISA) uses antibodies and a solid-phase enzyme immunoassay to detect the presence of a specific antigen in a liquid or wet sample. Antigens from the sample are immobilised on a solid support either non-specifically or specifically. A specific antibody is then applied over the surface so it can bind to the antigen. This antibody is linked to an enzyme or can itself be detected by an enzyme-linked secondary antibody. In the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a visible signal, most commonly a colour change in the substrate, which indicates the quantity of antigen in the sample.
When the presence of an antigen is analysed, the name "direct ELISA" refers to an ELISA in which only a labelled primary antibody is used, whereas the term "indirect ELISA" refers to an ELISA in which the antigen is bound by the primary antibody which then is detected by a labelled secondary antibody.
Radio Immunoassay Notes
This document discusses the history and development of radioimmunoassay (RIA) by Rosalyn Yalow, who won the Nobel Prize for her work. RIA involves labeling an antigen with a radioactive isotope, then using the specificity of the antibody-antigen reaction to separate bound labeled antigen from unbound antigen. This allows for quantification of antigens even at very low concentrations. The document outlines the principles, procedures, applications, advantages and disadvantages of RIA.
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BLO: Transferring the macromolecule from gel to membrane followed by detection on the membrane using antibody is k/a blotting
molecular methods used to identify and measure specific DNA, RNA and protein in complex biological mixtures.
It is the technique för
transferring DNA, RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis.
IMMUNO BLOTTING:
Immunoblotting techniques use antibodies to identify target proteins .
They involve identification of protein target via antigen-antibody (or protein-ligand) specific reactions.
The Southern blot is used for transferring DNA,.
The Northern blot for RNA
The western blot for PROTEIN.
The Eastern blot for PROTEIN, post-translational modifications (PTMS) .
WESTERN BLOTTING:
Principle:
Western blotting technique is used for identification of particular protein from the mixture of protein.
In this method labelled antibody against particular protein is used identify the desired protein, so it is a specific test.
Western blotting is also known as immunoblotting because it uses antibodies to detect the protein.
METHODOLOGY:
Extraction of protein
2. Gel electrophoresis: SDS PAGE
3. Blotting: electrical or capillary blotting
4. Blocking: BSA
5. Treatment with primary antibody
6. Treatment with secondary antibody( enzyme labelled anti Ab)
7. Treatment with specific substrate; if enzyme is alkaline phosphatase, substrate is p-nitro phenyl phosphate which give color.
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Proteases, glycosidases, antibody characterization tools, IdeS, IdeZ, mass spec reference protein digests, and new solutions for mass spectrometry analysis. Découvrez nos solutions pour vos analyses en spectrométrie de Masse.
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Immunoprecipitation (IP) - Creative Diagnostics
- 1. Creative Diagnostics Immunoprecipitation (IP) Overview and Technical Tips CDwww. creative-diagnostics.com
- 2. www. creative-diagnostics.com CD Introduction Immunoprecipitation (IP) is one of the most widely used antibody-based techniques. By targeting a known protein with a specific antibody, it may be possible to pull the entire protein complex out of solution. It is used to purify and enrich the protein of interest from a complex mixture. IP is an important step in many proteomics studies.
- 3. www. creative-diagnostics.com CD proteinprotein IP Immunoprecipitation Types protein Interaction partner CO-IP protein ChIP protein RIP
- 4. www. creative-diagnostics.com CD General Protocol Sample Preparation Pre-clear the Lysate Immunoprecipitation Elution
- 5. Harvest cells and wash with PBS Resuspend the cell pellet in ice-cold cell lysis Sonicate cells in ice bath Centrifuge and collect the supernatant Sample preparation Preparation of cell lysates www. creative-diagnostics.com CD
- 6. Wash the beads twice with PBS Dilute beads 1:1 with PBS Sample preparation Preparation of Protein A/G agarose beads www. creative-diagnostics.com CD It is recommended to use wide orifice pipette tips or tips with the end cut off when manipulating agarose beads to avoid disruption of the beads.
- 7. Pre-clear the cell lysates www. creative-diagnostics.com CD Pre-clearing the lysate is recommended to reduce the non-specific binding Add pre-washed Protein A/G agarose bead to the cell lysate. Incubate at 4 °C for 10 min with gently agitation. Centrifuge at 3,000 × g at 4 °C for 2 min and collect the supernatant
- 8. Immunoprecipitation www. creative-diagnostics.com CD Add relevant antibody to the pre-cleared cell lysate. And gently rock the mixture at 4 °C for 2-4 h or overnight. Add pre-washed Protein A or G sepharose beads slurry to capture the immunocomplex. Gently rock the mixture at 4 °C for 1–4 h. Collect the beads by low speed centrifugation at 2,000 × g, 30s at 4 °C. Wash the beads with cell lysis buffer (stringent) or PBS (less stringent) and collect the beads by low speed centrifugation at 4 °C.
- 9. Elution www. creative-diagnostics.com CD Protein can be eluted from the beads by denature buffer or non-denature buffer (low-pH glycine buffer). Increasing the salt concentration in elution buffer will help to elute protein. The eluent can be stored on ice for same day analysis or frozen at -80˚C for future analysis. SDS buffer: Harsh buffer can denature protein Glycine buffer: More gentle can dissociate protein of interest without eluting IP antibody
- 10. www. creative-diagnostics.com CD Analysis Analysis can be done using the following methods • SDS-PAGE • Western Blotting • Gel band Excision and sequencing • Mass Spectrometry • Scintillation counter or X-ray film for radioactive samples, etc.
- 11. www. creative-diagnostics.com CD Application • Isolate/detect proteins of interest. • Enrich the low abundant proteins. • Study protein-protein interaction and protein complexes • Identify unknown proteins in protein complexes
- 12. www.creative-diagnostics.com CD Thanks Creative Diagnostics For more info please contact us: Email:info@creative-diagnostics.com
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