All tags Epigenetics Introduction to ChIP principles and troubleshooting, webinar

Introduction to ChIP principles and troubleshooting, webinar

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This webinar covers the basics of Chromatin Immunoprecipitation (ChIP) including how to optimize a ChIP experiment.

ChIP is a very powerful technique that allows for the analysis of protein localization on DNA regions in the genome. Selective enrichment of a chromatin fraction using antibodies allows for the isolation of DNA regions and interacting proteins. Antibodies that recognize a protein or protein modification are used to determine protein localization and post-translational modification in the genome. The ChIP technique can be used in any area of research to further elucidate gene function and regulation in their native state.

ChIP webinar

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Highlighted points in this webinar are:

  • An introduction to chromatin and ChIP 
  • ChIP Protocols
  • Antibody selection
  • Troubleshooting tips and optimization
  • ChIP resources

Webinar transcript: 

Hi, welcome to Abcam's webinar on an Introduction to ChIP. This webinar is being recorded and will be available for viewing on the Abcam blog at a later date. Today the principal presenter will be Dr Karen Halls, our Scientific Support Manager. Karen completed her BSc Biology at the University of Birmingham, and a PhD from the University of Cambridge. Karen gained her PhD in the lab of Professor Tony Kouzarides studying SET proteins and novel histone modifications. Karen contributed to the Human Genome Sequencing Project and has extensive experience of ChIP. She joined Abcam's scientific support team in 2007. Joining Karen today will be Miriam Ferrer, a member of our new products team. Miriam completed her Biology degree at the University of Barcelona, followed by a PhD from the Free University in Amsterdam. During her PhD, Miriam worked on using the cisplatin sensitivity of Falconie anaemia cells for cancer therapy, and she expanded her experience on the DNA repair field by working on the BRCA1 during her postdoc at the LMB in Cambridge. Miriam joined Abcam in 2008.​

As mentioned previously, if you have any questions during the presentation they can be submitted at any time through the Q&A panel on the lower right hand side of your screen. The questions will be answered during the troubleshooting session at the end of the webinar. I will now hand over to Karen who will start the scientific talk on ChIP.

KH:    Thank you, Lucy. Welcome to our ChIP webinar. Thank you for taking the time to join us today, and I hope you will find the information useful. The webinar will be split into a variety of topics. First of all, there will be an introduction to chromatin and ChIP with background information about the technique. I will then run through the protocols step-by-step. There will be more information about selecting an antibody to use in ChIP. I will then discuss optimisation requirements with information on troubleshooting, and how problems can be resolved. In the final section, Miriam will give more information on protocol resources. Let's start by introducing chromatin and how ChIP can be used. The function of chromatin is to package DNA so it sits within the cell to strengthen DNA to assist with mitosis and meiosis, and help control gene expression. In eukaryotes chromatin is a complex of DNA, RNA and proteins. The major protein component is histones, although many other chromosomal proteins also play a prominent role.

A 147 base pairs of DNA is wrapped around the histone octamer to form the fundamental unit of chromatin: the nucleosome. This consists of two copies each of histones H2A, H2B, H3 and H4. Nucleosomes are compacted further to form the chromatin fibre by H1 and non-histone proteins. N- and C-terminal tails of the histone proteins extend from the nucleosome core, and a large number of amino acid residues can be conveniently modified with chemical groups and processes such as acetylation, methylation, phosphorylation, ubiquitination, citrullination. Cis-trans proline isomerisation results in a conformational change of the histone tail. These modifications function either by disrupting chromatin contacts, or by affecting the recruitment of non-histone proteins to chromatin. A number of enzymes have been identified, which catalyse the addition and removal of these modifications to histone proteins. Histone H3 is the most characterised histone protein, and the majority of these modifications exist in the end terminal tail.

The principle with ChIP is simple, it allows the selective enrichment of a chromatin fraction containing a specific antigen, and a specific DNA associated is identified. The most common purpose is to analyse the presence of a histone modification, or chromatin associated protein at genomic loci. Specific antibodies are used to determine the abundance of that antigen at one or more locations in the genome in vivo. Antibodies are used to pull out the proteins or protein modification of interest, and identify their localisation in the genome. I'm going to run through some examples to help illustrate this. ChIP can allow you to compare the enrichment of a protein, or protein modification at different loci. In this example histone H3K9 acetylation is being analysed across a small selection of active and inactive loci. The bar chart shows that this mark is enriched at the active genes analysed.

ChIP can also allow you to map a protein or protein modification across specific loci of interest. In this example the active RNA polymerase is being mapped across the gamma actin gene, with an antibody against phosphor serine-5 on RNA polymerase II. The target localises to the 5-prime end of the gamma actin gene, which is characteristic of this target. Finally, ChIP can also allow you to quantify a protein or protein modification at genomic loci over a time course. In this example, citrullinated H3 is being analysed at the inducible pS2 promoter. Further induced with oestrogen and samples taken every ten minutes, the cells from each time point are used for individual ChIP experiments with a specific antibody. The histone modification shows a cyclic presence at the promoter with highest levels of histone H3 citrullination at 40 minutes. There are two general procedures for performing ChIP experiments: native ChIP and cross-linking ChIP. The choice is dependent on your experiment or aims, and starting material. There are key differences between the two techniques.

In native ChIP, proteins are not cross-linked and native chromatin is the substrate. Fragmentation is achieved by micrococcal nuclease digestion, and using enzymatic digestion it is possible to obtain nucleosome-based resolution of approximately 150 base pairs. Native ChIP is only suitable for protein stably associated to chromatin; typically limiting this to histones and histone modifications. In cross-linking ChIP, proteins are cross-linked to DNA and formaldehyde is typically used, which is a reversible cross-linker. Chromatin is fragmented by sonication to generate random fragments of between 200 and 1,000 base pairs. As the proteins are cross-linked, histones, histone modifications and chromatin associated factors can be analysed. X-ChIP is standardly used at Abcam during antibody characterisation. There are a variety of pros and cons for each technique. In native ChIP, antibody specificity is predictable as the epitope is not affected by fixation. This also results in efficient immunoprecipitation as the antibodies are able to bind effectively to the target antigen. It is possible to fragment DNA to a nucleosome size of approximately 175 base pairs, giving higher resolution and more accurate mapping of the target antigen.

However, native ChIP is generally only suitable for histone proteins as chromatin associated factors are not tightly associated to DNA, and are likely to be lost during sample preparation. Using an enzyme will also result in some selective nuclease digestion, as the micrococcal nuclease will be favour some genomic sequences leading to unequal digestion. Nucleosome rearrangement can also occur as the nucleosomes are not fixed. Ultimately, care must be taken throughout the process as interactions are not stably fixed. Cross-linking ChIP is good for non-histone proteins that may bind weakly or indirectly to DNA, as cross-linking will fix these interactions. Cross-linking minimises nucleosomal rearrangements as interactions will stabilise, and there will be less variability between experiments. X-ChIP is preferable for yeast and other organisms where native chromatin is difficult to prepare. DNA is fragmented randomly by sonication. However, you may observe inefficient antibody binding due to epitope disruption, and it may be necessary to test a variety of different antibodies to find the best one.

Transient interactions may be fixed leading up to artifactual results. X-ChIP produces lower resolution maps as chromatin is prepared to larger fragment sizes, and the target localised to a larger region. Next, I will run through the protocol and detail what controls to include. The ChIP protocol is relatively straightforward. If you're performing an X-ChIP cells are treated to cross-link DNA and proteins. For both techniques a unicellular cell suspension is prepared in a lysis buffer. The chromatin is then fragmented by micrococcal nucleose digestion for N-ChIP, or sonication if performing X-ChIP. Antibodies are then used to immunoprecipitate the chromatin fragments where your target of interest is present. The subsequent DNA is then purified and analysed to determine the regions where the target is enriched relative to the input DNA. If performing X-ChIP, cross-linking is the first step and this is required to stabilise protein/protein and protein/DNA interactions. Histones themselves don't generally need to be cross-linked, but if your target protein is not specifically bound to DNA, ChIP will be less-effective without cross-linking.

The purpose is to fix the antigen to the chromatin binding site at fixed molecular interactions at a point in time. Formaldehyde generates reversible protein/protein links and allows you to study histone proteins and chromatin associated proteins. If the chromatin isn't fixed, then it's highly likely the associated proteins will be lost. A good starting concentration is 0.75 per cent of formaldehyde for cells in culture, or 1.5 per cent for tissues. This is added directly to the cells if using cell culture, or to a cell suspension after disaggregation of tissue is a starting material. The optimal time is between two and thirty minutes, the cells are then washed with ice-cold PBS and resuspended in lysis buffer. The next stage is to fragment the chromatin. This renders the chromatin soluble and determines the resolution of the assay. This means the extent to which you can fine map the location of a specific protein, the idle fragment side is between 170 and 1,000 base pairs. Start with a unicellular suspension in a lysis buffer. If performing X-ChIP the chromatin is fragmented randomly by sonication, and the optimal fragment size is between 200 and 1,000 base pairs.

It is important not to sonicate extensively as nucleosomes will become displaced from the chromatin. The fragment size will also always be slightly greater than the mononucleosome to minimise this. If performing N-ChIP, use micrococcal nuclease to digest the chromatin at 37° for five minutes and add EDTA to stop the reaction. Centrifuge the samples and the supernatant will contain smaller mononucleosome fragments. The pellet is resuspended in lysis buffer and dialysed to isolate larger fragments. One of the benefits of N-ChIP is that you can isolate mononucleosomes and oligonucleosome fragments; meaning you can associate the histone modification to the exact nucleosome. However, more care is required as it can be tricky to get right, and nucleosomes can rearrange during the enzymatic digestion. It is essential to optimise chromatin fragmentation. The next step is to perform an immunoprecipitation to purify the chromatin fraction that contains your target of interest. An antibody is used to isolate the protein and DNA complex. An antibody binding matrix is then used to pull down the entire complex. The fragmented chromatin sample is incubated with a primary antibody to a target of interest, and the antibody binding matrix overnight at 4° with rotation. The amount of antibody generally ranges from 1 to 10 micrograms.

Protein A or protein G agarose, sepharose, or magnetic beads are used for antibody binding. The exact matrix will depend on the isotype and species of your antibody. The following day the IP complex is then washed to eliminate any non-specific binding. The input should be set aside here, as this will represent the amount of chromatin used in each IP. The next step is to purify the DNA from the immunoprecipitated complex, and quantify where the target is bound. If samples have been cross-linked, then it is necessary to reverse the protein/protein and protein/DNA cross-links. The input is included at this stage and this will represent the total chromatin included in the IP that has not been incubated with antibodies. The total DNA will be purified. The protein/DNA antibody complex is eluted from the beads typically with 1 per cent SDS and 100 millimolar of sodium bicarbonate. If cells have been cross-linked, then the eluted DNA will be incubated with either proteinase K and RNase A, or sodium chloride for four to five hours at 65°. There are a number of variations for reversing the cross-links. If performing N-ChIP cross-link reversal is not required, but incubating with proteinase K, can sometimes enhance the DNA extraction. The DNA is then purified either using a PCR purification kit, or phenol chloroform extraction.

DNA analysis is required to identify the enrichment or localisation of your targets. The level of enrichment is always expressed as a ratio of bound or immunoprecipitated DNA over input. This is because the enrichment of the target is dependent on many factors, such as antigen accessibility, antibody affinity, the specific IP conditions, the starting material or sample quantity. Therefore, in all the analysis methods described, the amount of DNA using specific antibodies is always compared to the input, which is the total amount of DNA in the starting sample. There are three key methods of DNA analysis: the protein or protein modification can be mapped at specific regions using ChIP (PCR) or across the entire genome using ChIP-chip or ChIP-seq. I shall now discuss these in more detail. In ChIP (PCR) isolated DNA is quantified by real-time PCR typically using TaqMan or SYBR Green technology to amplify, and simultaneously quantify a target DNA molecule by measuring changes in fluorescence. This allows the analysis of a specific region in multiple samples and can be quicker, and more cost-effective when compared to ChIP-chip or ChIP-seq. The level of fluorescence is proportional to the amount of target DNA indicating antibody binding, and subsequent presence of the target protein.

In this example, ab8898 which detects histone HG3 trimethyl K9, has been used in ChIP on U20S cells. DNA primers specific to active, inactive and heterochromatic genes have been used in a PCR where the isolated DNA and input DNA is the template. This will provide actual DNA amounts of the target DNA in both the input and ChIP samples. The graph shows the amount of DNA relative to the input, and you can see that there is a greater amplification and enough small DNA present of the heterochromatic genes. This indicates that the histones modified with histone H3 trimethyl K9 are enriched at these genes. ChIP-chip employs microarray technology to give high resolution genome-wide maps of protein, and protein modifications. Purified DNA is labelled with fluorescent dyes using ligation mediated PCR. The fluorescently labelled DNA is then applied to the microarray, and after subsequent image analysis the enrichment of the target protein relative to the input is recorded at each genomic locus.

In the example shown, a GSP tagged protein has been localised in a saccharomyces cerevisiae microarray. The anti-GSP antibody ab290 has been used in ChIP and the subsequent DNA has been applied to the yeast microarray. The sample with the anti-GSP antibody is shown in green, and then untagged control is shown in purple. DNA levels are clearly elevated at the promoters of two specific genes in the array, indicating that the target protein is enriched at these regions. In ChIP-seq the isolated DNA is directly sequenced using next-generation sequencing to generate a genome-wide profile. It combines ChIP and direct sequencing for genome-wide analysis of antigen distribution. An immunoprecipitated DNA is mapped to the genome and enables large amounts of DNA to be sequenced in a matter of days. In this example, an antibody against RNA polymerase II phosphorylated at serine-5, has been used in ChIP and the isolated DNA directly sequenced. The resulting profile shows peaks in DNA levels at two gene promoters, indicating that serine-5 phosphorylation of RNA polymerase II is enriched at these two promoters. This is only a snapshot of one generic region, but you could feasibly look at any region within the genome when using ChIP-seq.

Several controls should be used in each ChIP experiment to ensure the procedure is working as expected. Negative controls would give the background level of the experiment. Use beads-only or beads with an isotype match control immunoglobulin. For example, if you're using a rabbit IgG primary, ab27478 is a suitable isotype control as it is also a rabbit IgG, but is not specific to a target protein. Positive and negative control loci are regions where you know the protein or modification is present, and where it is absent. This should be reviewed in the whole genome map, or specific primers should be designed and tested in real-time PCR. This will tell you whether the observed enrichment is specific. Some antibodies will give non-specific enrichment, and this will be determined by a high signal in the positive and negative control loci. The no-template control should always be included in the PCR, it will help you spot any contamination, as there should be no amplification in these samples. Finally, use a positive control antibody against the target where its localisation is well-characterised. Use primers or analyse the region of the genome where the positive control is found to ensure it is enriched, this will confirm that the procedure is working well.

If studying active regions, you can use antibody specific for histone H3 trimethyl K4 such as ab8580. This is enriched on actively transcribed regions. If studying inactive regions you could use a marker against inactive genes, such as histone H3 trimethyl K27 ab6002. Both are good controls to ensure each step of the experiment is working, however, if these histone modifications are not present at your region of interest, then they will not be suitable so the controls must be specific. Here are ChIP results for ab8580 raised against H3 trimethyl K4 to illustrate how controls are used. The beads only controlling yellow is a negative control and it gives you the background level, and should be significantly lower than the signal with the antibody. A number of active genes such as GAPDH, LPL30 and ALDOA have been included as positive control loci, as the histone modification is found here. Inactive genes such as MYO-D, SERPINA and AFM are the negative control loci, as the histone modification does not localise the inactive genes. The no-template control is not shown here but is included in the PCR, and no amplification is observed. This antibody is a good positive control antibody, it is known that the histone modification is enriched at active genes, therefore, this would be a great positive control antibody for the localisation of potential chromatin associated proteins, or another histone modification at active genes.

Next, I will discuss antibody selection. In ChIP an antibody is used to capture specific DNA associated proteins and success is dependent on the quality of the antibody. Many ChIP grade antibodies are available, but if this is not the case here are some tips on how to select a suitable antibody. Antibodies should be specific and efficient, even if it works in IP it may not work in ChIP. This is largely due to the effect of cross-linking as specific epitopes can be generated and lost. Ideally, antibodies should be affinity purified to remove any cross-reactivity. Histone modification antibodies should be tested in Western blot analyser for cross-reactivity with other histone modifications. In this example ab8898, which detects histone H3 trimethyl K27, has been tested against a panel of peptides in a competition experiment to check for any binding with other histone modifications. In this case, there is minimal cross-reactivity to the majority of peptides tested. There is slight cross-reactivity with H3 trimethyl K27, and this is expected as the amino acids surrounding K9 are the same as K27. Cross-reactivity can also be tested in an ELISA peptide array.

In this example, ab8580 has been tested in a peptide array against peptides that correspond to a number of different histone H3 modifications. The histone modifications represented here are mono- di- tri- K4, mono- di- tri- K9 and mono- di- tri- K27. Six dilutions of each antibody are printed onto the peptide array in triplicate, and results are averaged before being plotted onto the graph. Results show strong binding to the histone H3 trimethyl K4 peptide, which is ab1342, indicating that this antibody specifically recognises the histone modification it was raised against. Immunoprecipitation, immunohistochemistry and immunocytochemistry are good indicators of success in ChIP. In these techniques, the epitope is recognised and accessible in its native conformation, and within a complex context. In Western blot the epitope is denatured, so the experimental context is different and the epitope will be presented differently. Therefore, if the antibody works in IP, IC or ICC then it is more likely to also work in ChIP.

There is debate as to whether polyclonal or monoclonal antibodies are better for ChIP. Quite simply there are pros and cons for both. There is an increased probability that a polyclonal antibody will work in ChIP, as it contains a mixed antibody population. Therefore, if one of the epitopes is masked and one of the antibodies cannot bind, it is highly likely another antibody in the population will recognise and bind a slightly different epitope. This is in contrast to a monoclonal antibody, which is derived from a single clone and this will recognise a single epitope. If this epitope is altered or masked the antibody will not bind, and the antibody will not work. However, monoclonal antibodies should show less batch-to-batch variation, as it will recognise the same epitope between batches. Whereas polyclonal antibody populations will vary between batches and epitope recognition may change. Ultimately, there are some excellent poly- and monoclonal antibodies and this should not be a crucial deciding factor as to which antibody to choose. Optimisation may be necessary to obtain optimal results and in the next section I shall give advice on which areas to place close attention to.

Formaldehyde is used as a cross-linking agent to fix protein/protein and protein/DNA interactions. It may be necessary to add glycine to quench the formaldehyde if you find the samples are fixed for too long, or alternatively you could reduce the fixation time. The fixation time can range from two to thirty minutes, but ten to fifteen minutes is a good starting point. This may need to be longer for tissue samples, as permeation levels will be slower. Excessive cross-linking results in reduced antibody binding and an inefficient immunoprecipitation, due to a reduction in antigen accessibility. Epitopes are altered when new epitopes are being generated or lost. Excessive cross-linking also results in a decrease in protein binding. Insufficient cross-linking results in protein dissociation, as there is no stable association between DNA and proteins. It is best to start with standard conditions and optimise if necessary, performing a time course with different concentrations of formaldehyde.

It is essential to optimise DNA fragmentation and the idle fragment size is between 150 and 1,000 base pairs. Sonication conditions are affected by foam and cell density, as this would affect energy transfer within the solution. Cross-linking affects the density of the solution. Some cell types and cell lines are easier to sonicate than others, therefore, it is necessary to optimise chromatin fragmentation for each cell type used, and then keep the parameters constant for reproduce of all results. In the example on the right hand side, a sonication time course experiment is shown. U2OS cells have been sonicated for five, ten, fifteen and twenty minutes. The cross-links reversed, DNA purified and DNA resolved on a 1.5 per cent gel. The fragment size decreases during the time course and the optimal fragment size is at 15 minutes. At twenty minutes it is likely that during the sonication process proteins have been lost in the DNA due to excessive sonication; this will release nucleosomes from the DNA.

Similarly, when digesting with micrococcal nuclease it is important to perform a time course with different concentrations and incubation times. High concentrations and extended digestion times may over-digest a chromatin leading to a subnucleosome or particles. When using new enzyme stocks and samples, optimisation will be necessary as there will be some variation. The optimal antibody concentration should be determined for each antibody to improve the signal to noise ratio. Use approximately 1 to 10 micrograms of antibody per 25 micrograms of chromatin; 5 micrograms is a good starting point. In the example shown, ab12089 and anti-TBP antibody has been tested at amounts of 2 micrograms and 10 micrograms. TBP localises the active genes; when using 2 micrograms the signal is very similar to background with little difference between active and inactive genes. However, when using 10 micrograms there is a significant increase in signal, especially as active genes, therefore, in these ChIP conditions, 10 micrograms of this antibody should be used.

It is important to note that the amounts used should be optimised by each user, as 2 micrograms may be sufficient in another user's experimental conditions, and there are no standard rules that will work for everyone. Different antibodies have different affinities to their target, therefore, you may need to experiment with different salt concentrations in the wash steps. In most protocols, the antibody protein DNA complex is washed three times with a buffer containing 150 millimolar of sodium chloride, then a more stringent wash buffer containing 500 millimolar of sodium chloride is used. Though affinity antibodies may disassociate from target proteins when using stringent wash buffers, so it is worth experimenting with a lower salt concentration to ensure that specific antibody binding is not compromised. In this example this shows ab818, another TBP antibody. When using 500 millimolar of salt in the final wash solution, the difference between the specific antibody ChIP and beads-only control, is relatively low with may be six times enrichment.

However, reducing the final wash salt content to 250 millimolar leads to an increase in specific antibody binding, but without a subsequent increase in background. The stringency should be as high as possible to give a high signal to noise ratio, but without compromising specific antibody binding. Next, I will highlight some common problems and how these can be resolved. You may observe high background in non-specific antibody controls where beads-only or an isotype control is being used. This may be due to non-specific binding to protein A or G beads. Including a pre-clearing step may help. The live sample is mixed with beads alone for one hour and removed prior to adding the antibody. Any non-specific binding to the beads should be eliminated. The ChIP buffers might be contaminated, therefore, prepare fresh lysis buffers and wash solutions. Some protein A or gBs can give higher background levels. Try different beads to find those that provide the cleanest results with low background in a non-specific control. Consider switching from agarose, sepharose to magnetic beads, as this may also reduce background binding to the beads.

You may observe low resolution with high background across large regions, this occurs if the chromatin fragment size is too large. DNA fragmentation should be optimised when using different cell types, as both sonication and enzyme incubation times can vary. The DNA fragment size should be no larger than 1,500 base pairs. Consider increasing the sonication or digestion times. You may observe low signal across all samples; the chromatin fragment size may be too small. Reduce the sonication and digestion time to increase fragment size to greater than 500 base pairs. Sonication to a smaller size can displace nucleosomes as internucleosomal DNA becomes digested. The cells may not have been effectively lysed. Reaper generally give good results, however, slight variations with increased detergent levels may improve the signal. The cells may be cross-linked for too long. Excessive cross-linking can reduce the availability of epitopes and thus reduce antibody binding. If performing X-ChIP, cross-link formaldehyde for a reduced amount of time, and wash cells with PBS. Cells may need to be treated with glycine to quench the formaldehyde.

You may need to increase the amount of starting material, as the levels may be insufficient for ChIP. Standardly, 25 micrograms of chromatin is used per IP, but this may need to be increased. There may be insufficient antibody in the IP; 3 to 5 micrograms of antibody is a good starting point, but this could be increased to 10 micrograms if no signal or weak signal is observed. The specific antibody binding might be eliminated by washing; don't use higher than 500 millimolar of sodium chloride in the wash buffers, as this may be too stringent and eliminate specific antibody binding. Consider using 250 millimolar of sodium chloride in the final wash buffer. The target might not be enriched at the region of interest, be sure to include a positive controlled antibody to confirm the procedure is working well. The wrong antibody affinity beads may have been used and the antibody not isolated. It's essential to use an affinity matrix that will bind your antibody of interest. Protein A and G are bacterial proteins that bind various classes of immunoglobulins with varying affinities, so it's essential to check compatibility. Start using a mix of protein A and protein G that have been coupled to sepharose or magnetic beads, if there is no binding preference for the isotype.

The antibody might not be suitable for ChIP. Consider trying another antibody as the epitope may not be accessible, or may be presented differently. If performing X-ChIP you could try N-ChIP, as the epitope will be available in its native form. If performing N-ChIP, the target protein may have a weaker DNA affinity, or may not be in close proximity of the histone. Cross-linking might be required for an efficient IP. There may be problems with PCR amplification on immunoprecipitated DNA, you may see a higher signal in all samples after PCR, including the no-template control. This may be due to contamination of real-time PCR solutions, so prepare new solutions from stocks. You may see no DNA amplification in samples, be sure to include standards and input DNA to confirm that the primers are working well, and prepare PCR primers to several regions. Certain areas of the genome will purify better than others, and some nucleosomes may rearrange during enzymatic fragmentation. I would now like to handover to Miriam who will give more information about protocols and our troubleshooting guidebook.

MF:    Thanks Karen. Hello, I would like to take this opportunity to tell you a bit more about some of the resources and products that Abcam has available for epigenetic research, with a special focus on ChIP. As most of you probably know, our Beginner's Guide to ChIP Guidebook is an ideal resource tool for everyone using the ChIP technique. It contains advice and troubleshooting tips from top researchers, including our own R&D scientists, in a step-by-step guide through the experiment. Some of the topics that Karen has discussed are also pointed out in this book. The guidebook can be downloaded from our website, but you can also request a hardcopy to be sent to you by sending your request to the mailrequest@abcam.com email address. We have recently added two new applications, and they are our at Abpromise guarantee. The RIP which stands for RNA Immunoprecipitation, and CLIP which stands for UV Cross-link and Immunoprecipitation. These are antibody-based techniques that are used to study RNA and protein interactions.

If you would like to know more about these applications, please check out our protocol page at abcam.com/protocols, or our epigenetics microsite at abcam.com/epigenetics. If you have any questions regarding any of the topics discussed today or on any Abcam products, please feel free to contact our scientific support team. They will be very happy to help you with any query you might have. To those of you who are located in the US, Canada or South America please contact our US team. If you are in Hong Kong, China or Asia please contact our Hong Kong team. If you are located in the UK or Europe, please contact our UK team. If you are in Japan, please contact our Japanese team. I would like to highlight as well that we have multiple language support, so please do not hesitate to contact us. abcam.com/epigenetics, as I've just mentioned before, is our new epigenetics microsite, this is a one-stop shop for all the topics related to the genetics. In this microsite you can find the latest information on products, protocols and upcoming meetings all related through epigenetics.

If you're planning to use large quantities of one product, you might want to look into buying it in bulk. Bulk-buying will help you to save some money, and to minimise the viability in your experiment. If you want to know more information about our all-bulk options, please contact our sales team at sales@abcam.com. I would like to focus now on our new range of Abcam epigenetic kits, the EpiSeeker range. These kits have been designed with the researchers in mind, so that you can spend less time doing the experiment and actually having more time to think about the design and the outcome you want from the experiment. Although our EpiSeeker range includes a lot of different products, I will just mention today our range of ChIP kits. Just to point out that all our EpiSeeker ChIP kits are for cross-link ChIP and cannot be used for native ChIP. Our one step and plant ChIP kits have been optimised for mammalian and plant DNA, respectively. These kits in particular don't contain an antibody in the kit, and are therefore adaptable to any target. The EpiSeeker range also includes kits optimised for methylated or acetylated histone modifications, whether your starting materials are cells or tissue. We also have kits for immunoprecipitation of methylated DNA on which I will elaborate a bit further on. More details on these kits and other EpiSeeker products can be found at the page pointed there, abcam.com/EpiSeeker.

You might be wondering what are the advantages of using these EpiSeeker ChIP kits instead of following the conventional ChIP method? To start with, the reaction takes place on a 96 well plate, so they are very easy to standardise and to perform in a high throughput assay. It only takes five hours in comparison to the two days you have to spend on if you follow the conventional protocols. It contains all the necessary reagents for the ChIP reaction, except for the cross-linking step, so everything is contained in your kit. Except for the general ChIP kits, all our ChiP kits contain a preselected ChIP grade antibody which has been optimised for the assay. Last, but not least, once you get your precipitated DNA it can be used straightaway for whichever downstream process you might want to. As mentioned previously, we offer methylated DNA monoprecipitation kits. These two kits contain specific antibodies to specifically enrich methylated and hydroxymethylated DNA specifically. These modifications simply play an important role on differential gene expression, and therefore it is very important to have the proper tools to investigate their functions. Our immunoprecipitation kits are also very useful to complement all the DNA methylation experiments, such as the bisulfite modification kits, where that is not possible to differentiate between a 5-methyl cytosine and 5-hydroxymethyl cytosine.

I just wanted to finish by thanking you all for attending this seminar, and for all webinar attendees we have at the moment a special offer with a 25 per cent discount on any of the EpiSeeker ChIP kits. Moreover, if you provide us with some feedback data on these ChIP kits, you can get 25 per cent off any ChIP grade primary antibody. After this meeting, you will be directed to our website where you can find more information about this special offer, plus a downloadable copy of this webinar. Without further delay, I'll pass you back over to Karen who is ready to answer the questions we've received during the webinar, and thank you very much for your attention.

KH:    Thank you, Miriam. We've received quite a lot of questions during the webinar, and I'll answer some of these now. All unanswered questions will be answered by email after this webinar. One of the questions comes in from Maria who asks: Is it necessary to use glycine to quench the formaldehyde? No, it isn't necessary to use glycine to quench formaldehyde. It is definitely very useful to do that when you're using, say, tissues where your sample will be a lot thicker and the penetration might be more difficult. You may then cross-link for a longer amount of time, because you're not able to wash that all away. So I would definitely recommend it for unusual cell types, i.e. if you're not using cell culture or cell suspensions. There's another question from Aaron Dam asking about MNase digestion, and whether that can be used in cross-linking with ChIP? Yes, it is possible to use enzymes to digest your chromatin after you've been cross-linking. It is more difficult, because obviously the proteins and the DNA will be cross-linked, so it might be more difficult to penetrate. But it's definitely possible to do that; it might take a little bit of optimisation and it's definitely worth playing around with that to get the conditions right.

There's a question from Sallem asking which sonicator would you recommend for X-ChIP? In my experience, the sonicating water baths are better because they're more reproducible. You have little foaming than what you would do if you were using a sonicating probe. One thing to bear in mind, and it's very important to keep your sample cool; using ice in the water bath will help with this. So make sure that you keep your sample cool and you only do it for short bursts of time, so rather than sonicating for five minutes take breaks in between that; so sonicate for 30 seconds and then rest for 30 seconds, so do that intermittently. There's another question from Jens: Is it possible to perform ChIP with tissue samples? Yes, it is. What you just need to be careful of, or to make sure that you do is to grind the tissue so that it becomes a unicellular suspension. As I mentioned with the quenching, you might also need to cross-link for a little bit longer, or use an increased amount of formaldehyde for that. There's a protocol within the protocol's book that Miriam mentioned, that has more information about how to prepare your samples. But once you've prepared them and you've got a suspension* you can continue with the standard X-ChIP protocol, so that's definitely possible.

There's a question also, similarly, from Nina, actually, about using plant samples for ChIP, so is it possible to also use that as a sample type? Yes, again, that is also possible, you use seedlings for that and grind the material with a pestle and mortar to disaggregate the sample. Use a series of extraction buffers sonicating and continue with a standard ChIP protocol. We've also got a protocol for that within our protocol's book, and also a ChIP kit for plant material, so that could be a point of call with that's your starting material. There's a question here from Jan about controls, so if there is no IG control available for your primary antibody, for example, if it's something unusual then what control should I use? [Break in recording] that you can use an IG control, you can also use a beads-only control and that will give you the background of your assay. You can also use an antibody of the same species or isotype that is raised against something that's irrelevant. For example, an expression tag that won't be in your sample, just to give you some level of background and something to compare that to, so that's something that you could use.

There's a question from Todd about whether it's possible to use whole antiserum antibodies? Yes, you can use whole antiserum antibodies, obviously you have to be very mindful that you will get, or you may get higher background with these antibodies, because there'll be non-specific antibodies within the solution. It's worth testing, if that's the only option that you have available. You could also consider purifying the whole antiserum to give the IgG fraction, so it's a more pure product. You can use protein A or protein gBs to do that. So that's definitely worth giving a try, so there's no other ChIP antibodies or affinity purified antibodies available. Finally, because we're not able to get through them all, this is the final question that I'll cover today in the webinar, which is from Joe asking if an antibody works in ChIP would it also work in ChIP-seq? Yes, it will and the procedure is pretty much the same, it's just the output and the analysis is different. So if an antibody is tested in ChIP and is suitable for that, then it will also give you DNA that will be suitable for ChIP-seq, so yes. I'm going to handover to Lucy, thank you for your time and attention again today, and we will get back to any questions.

LP:    Hello, thanks Karen. On behalf of Abcam, I would like to thank you for attending this webinar and I hope you found it informative and useful for your work. For those whose questions were not answered, our scientific support team will contact you shortly with a response to your question. The webinar just given is available for download as a PDF. When you log-off from the webinar you will be redirected to a webpage where a PDF of the presentation can be found, along with information about the exclusive ChIP kits and antibody promotion, which is available to all webinar participants. If you have any questions about ChIP or have a scientific enquiry, as mentioned before, please contact our scientific support team who will be very happy to help you, and they can be contacted at technical@abcam.com. If you have any webinar-related questions, please contact the Abcam events team at events@abcam.com. Finally, we always welcome feedback on our webinars and would be grateful if you could take a minute to complete the short feedback survey, which will pop up on your screen when you log-off from the webinar. I hope we can welcome you to a future webinar, and thank you again for attending and good luck with your research.