1. The signal istoo low
Chromatinfragments too small: to get less than 500bp chromatin fragments, no need forsonication. The too small fragment will be nucleosome mistaken for nucleosomeDNA and be digested. If end-chromatin immunoprecipitation, usually enzymaticdigestion method to get the desired size of chromatin fragments.
Cross-linkedChromatin immunoprecipitation cross-linking time is too long: cross-linked withformaldehyde for 10-15 minutes,
then washed with phosphate buffer, followed bythe need to add glycine to stop the role of formaldehyde. Over-crosslinking mayreduce the binding of the epitope and thus decrease the binding of theantibody.
Blocked bindingof specific antibodies: sodium chloride concentration in the wash buffer shouldnot exceed 500mM, or the concentration is too high will impede the specificantibody binding
Some MAbs arenot suitable for cross-linked chromatin immunoprecipitation: epitopesrecognized by MAb may be masked during cross-linking and prevent siterecognition. Polyclonal antibodies are recommended to recognize multipleepitopes, thus increasing the chance of immunoprecipitation of the targetprotein.
Misdirectedantibody affinity beads: Proteins A and G are bacterial-derived proteins thathave different affinities for different immunoglobulins. Please use a specificaffinity matrix.
There are notenough antibodies in the target area: There are no epitopes in the target area.Positive control antibody should be used to confirm the correct operation.
2.Immunoprecipitation of DNA when PCR amplification problems
A very highsignal appeared after the reaction, including negative control without atemplate: the reagents used for real-time PCR were contaminated. It isadvisable to use stock solutions to prepare solutions freshly
No DNAamplification in the sample: Use standard control/sample DNA to confirm thatthe primers in the sample are valid
3. combined withthe area too large background too high resolution is too low
Fragments toolarge: for different cell lines, DNA fragments should be optimized for size.Ultrasound time and enzyme incubation time should be adjusted. Do not recommendDNA fragments more than 1.5kbp. If enzymatic digestion of chromatin, will getthe 175bp size of a single nucleotide enzyme.
Fournon-specific antibody control background appears too high
No SpecificBinding to Protein A or G: Precleaning should be performed by mixing the lysedsample with magnetic beads for 1 hour before addition of the antibody, and thenseparated for use. Some protein A or G magnetic beads themselves produce highBackground: Some protein A or G beads have a high background. To find asuitable supplier, the product provided should have the lowest background,cleanest result in a nonspecific control sample
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