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Premium member Presentation Transcript Prepared by: mahmoud w. qandeel: Prepared by: mahmoud w. qandeel Chromatin immunoprecipitationSlide 2: Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins. Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point in the procedure. Types of immunoprecipitation Individual protein Immunoprecipitation (IP) Protein complex immunoprecipitation (Co-IP) Chromatin immunoprecipitation (ChIP) RNA immunoprecipitation (RIP) Tagged proteins What is the immunoprecipitation ?: Chromatin immunoprecipitation (ChIP) is a method used to determine the location of DNA binding sites on the genome for a particular protein of interest. This technique gives a picture of the protein-DNA interactions that occur inside the nucleus of living cells or tissues. ChIP can be used to determine whether a transcription factor interacts with a candidate target gene and is used with equal frequency to monitor the presence of histones with post-translational modifications at specific genomic locations. The in vivo nature of this method is in contrast to other approaches traditionally employed to answer the same questions. Chromatin immunoprecipitation (ChIP): The first chromatin immunoprecipitation (ChIP) assay was developed by Gilmour and Lis (1984 - 1986) as a technique for monitoring the association of RNA polymerase II with transcribed and poised genes in Escherichia coli and Drosophila .Slide 5: The principle of that assay is that DNA-binding proteins (including transcription factors and histones ) in living cells can be cross-linked to the DNA that they are binding. By using an antibody that is specific to a putative DNA binding protein, one can immunoprecipitate the protein-DNA complex out of cellular lysates . The principleSlide 6: Overview: Crosslink cells with formaldehyde. Shear DNA by : enzymatic digestion or sonication . Add antibody to a protein involved in transcription. Precipitate antibody and everything attached Heat to release DNA. Analyse DNA with PCR or microarrays The techniqueSlide 7: Cross-linking of DNA and proteins is often required to stabilize their interactions before analysis. ChIP can be performed in two different ways depending on whether you opt to cross-link your chromatin sample: if you cross-link your sample then the technique is termed “X-ChIP,” otherwise it is referred to as “N-ChIP.” The aim of cross-linking is to fix the antigen of interest to its chromatin binding site. Histones themselves generally do not need to be cross-linked, as they are already tightly associated with the DNA. Cross-linking: Other DNA binding proteins that have a weaker affinity for DNA or histones may need to be cross-linked. This holds them in place and avoids protein dissociating from the chromatin binding site. Use formaldehyde, as the links it forms are reversible. UV cross-linking is not appropriate as it is irreversible. Cross-linking is a time-critical procedure. Cross-linking should generally only be carried out for a few minutes. Excessive cross-linking can lead to a decrease in the amount of protein bound to the DNA, reduction in the availability of epitopes /changes in epitopes for antibody binding and, in turn, reductions in the material bound/antigen availability in your sample.Slide 9: Fragmentation of the chromatin is required to make interactions accessible to antibody reagents. To fragment chromatin, you can either sonicate it or digest it with using micrococcal nuclease. Which method you choose will largely depend upon the type of ChIP experiment being performed. Shearing of the DNASlide 10: Enzymatic digestion, with micrococcal nuclease, should be sufficient to fragment your sample for performing N-ChIP. N-ChIP does not call for cross-linking and so there will be no potential affects on the enzyme accessing its target. Using the enzymatic technique it is possible to generate single monosomes (~175 base pairs), providing the highest resolution in ChIP. Nucleosomes are dynamic and without cross-linking they may rearrange during the enzymatic digestion. This is a potential problem if you wish to map areas of the genome, and suitable controls must be used to monitor any changes N-ChIP with enzymatic digestionSlide 11: Typically, sonication is necessary for X-ChIP as formaldehyde cross-linking restricts the access of enzymes such as micrococcal nuclease to their targets, meaning that enzymatic digestion will normally be inefficient on cross-linked samples. Sonication is generally believed to create randomly sized DNA fragments, with no section of the genome being preferentially cleaved. The fragments created by sonicating , which average 500–700 base pairs (2–3 nucleosomes ), are typically larger than those created via enzymatic cleavage. The size of the fragments that are created directly affects the resolution of the ChIP procedure; fragments up to 1.5 kb resolve well for most purposes in ChIP. X-ChIP and sonicationSlide 12: Advantages Predictable and testable antibody specificity Efficient precipitation of DNA and protein High resolution (175 bp / monosomes ) Disadvantages Nucleosomes may rearrange during digestion Not useful for non- histone proteins. Selective nuclease digestion may bias input chromatin. Not useful for non- histone proteins Advantages Good for non- histone proteins binding weakly or indirectly to DNA Cross-linking minimizes nucleosome rearrangements Good for organisms where native chromatin is difficult to prepare (e.g., yeasts) Disadvantages May be inefficient antibody binding due to epitope disruption Fixes transient interactions to give a false picture of steady state levels. Lower resolution chromatin preparation by sonication Difficult to enzymatically digest cross-linked DNA N-ChIP X-ChIP Comparison between N-ChIP and X-ChIPSlide 13: Antibodies are used in ChIP to capture proteins and the interacting DNA. Antibodies used for ChIP should ideally be fully characterized. Characterizing antibody specificity using peptide competition in western blot is recommended for N-ChIP. Ideally, specific antibodies for ChIP should be affinity-purified; however many laboratories use sera as their antibody source and then overcome background problems that may arise with stringent buffers. A polyclonal antibody is preferable to a monoclonal,within a polyclonal antibody population there will be a number of antibodies that recognize different epitopes . Antibodies for ChIP: At this point the immunoprecipitation is performed resulting in the purification of protein-DNA complexes. The purified protein-DNA complexes are then heated to reverse the formaldehyde cross-linking of the protein and DNA complexes, allowing the DNA to be separated from the proteins.Slide 15: The identity and quantity of the DNA fragments isolated can then be determined by PCR. The limitation of performing PCR on the isolated fragments is that one must have an idea which genomic region is being targeted in order to generate the correct PCR primers. This limitation is very easily circumvented simply by cloning the isolated genomic DNA into a plasmid vector and then using primers that are specific to the cloning region of that vector. Alternatively, when one wants to find where the protein binds on a genome-wide scale, a DNA microarray can be used (ChIP-on-chip or ChIP-chip) allowing for the characterization of the cistrome . As well, ChIP-Sequencing has recently emerged as a new technology that can localize protein binding sites in a high-throughput, cost-effective fashion. DetectionSlide 17: ChIP has the advantage of measuring protein DNA interactions of promoter regions with protein complexes in the natural genomic state. Moreover, the ChIP assay can directly provide information on the histone acetylase status of specific chromatin regions, and be used to study non- histone proteins, even ones that do not directly bind DNA. With X-Chip, it also minimizes the chances of chromatin rearrangements during preparation and precipitation It can be used with gene-specific primers for increased sensitivity or with a specific probe, slot blot hybridization, for increased specificity. T he AdvantagesSlide 18: Dificulties with using ChIp will include, the danger that the cross-linking step may fix interactions that are of minor functional sugnificance , precipitation us often very inefficient, and purifying cross-linked DNA-protein usually requires isopycnic centrifugation, a prolonged and expensive procedure. A large number of cells are usually required and the data represents an average of potentially multiple functional states in the cell population. In addition, negative results do not necessarily mean that a given factor is not associated with the site. Certain antibodies are poor in immunoprecipitating crosslinked chromatin and lack of crosslinking can results from inaccessibility of factors in large complexes or biochemical properties of proteins that decrease cross-linking efficiency. The Disadvantages You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.