ChIP Troubleshooting Tips
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1. Troubleshooting tips ChIP abcam discover more High background in non specific antibody controls Non specific binding to Protein A or G beads Include a pre clearing step whereby the lysed sample is mixed with beads alone for 1 hr and removed prior to adding the antibody The ChIP buffers may be contaminated Prepare fresh lysis and wash solutions Some Protein A or G beads give high background Some Protein A or G beads can give high background levels Find a suitable supplier that provides the cleanest results with low background in the non specific control Low resolution with high background across large regions DNA fragment size may be too large DNA fragmentation should be optimised when using different cell types Both sonication times and enzyme incubation times can vary We would suggest a DNA fragment size of no larger than 1 5 kbp If chromatin is being digested using enzymes mononucleosomes 175 bp can be obtained Low signal The chromatin size may be too small Do not sonicate chromatin to a fragment size of less than 500 bp Sonication to a smaller size can displace nucleosomes as intra nucleosomal DNA becomes digested If performing N ChIP enzymatic digestion is generally sufficient to fragment chromatin If performing X ChIP the cells may have been cross linked for too long Cross link with formaldehyde for 10 15 mins and wash well with PBS Cells may need to be treated with glycine to quench the formald2. ehyde Excessive cross linking can reduce the availability of epitopes and thus reduce antibody binding Not enough starting material We would suggest using 25 ug of chromatin per immunoprecipitation Not enough antibody included in the immunoprecipitation We would suggest using between 3 5 ug of antibody in the first instance This could be increased to 10ug if no signal is observed Specific antibody binding is being eliminated Do not use higher than 500 mM NaCl in the wash buffers as this may be too stringent and remove specific antibody binding Cells are not effectively lysed We would suggest using RIPA buffer to lyse cells The composition can be found in our X ChIP protocol No antibody enrichment at region of interest The epitope is not found at the region of interest Be sure to include a positive control antibody to confirm the procedure is working well e g H3K4me3 H3K9me3 antibody at active inactive promoters N ChIP is not suitable X ChIP may be more suitable when analysing proteins that have either a weaker DNA affinity or are a long way from DNA Cross linking may be required to stop proteins dissociating from the DNA Histones are tightly associated therefore N ChIP can be performed when studying histones Discover more at abcam com technical Monoclonal antibodies may not be suitable for X ChIP The epitope may have become masked during cross linking thus preventing epitope recognition We would suggest using polyclonal an
3. tibodies that will recognize multiple epitopes as there is an increased chance of immunoprecipitating the protein of interest The wrong antibody affinity beads were used Protein A and G are bacterial proteins that bind various classes of immunoglobulins with varying affinities Use an affinity matrix that will bind your antibody of interest We would suggest using a mix of Protein A and protein G that have been coupled to sepharose Problems with PCR amplification on immunoprecipitated DNA High signal in all samples after PCR including no template control Contamination in real time PCR solutions we suggest preparing new solutions from stocks No DNA amplification in samples Be sure to include standard input DNA to confirm that the primers are working well Discover more at abcam com technical
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