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It is known that c-Jun and fos dimerize to form AP1 factor that binds to a sequence on DNA containing PyPuGACGTCNNNNGAGGTCPyPU. In esophageal cancer cell lines there is no expression of the fos gene. However we still see expression of genes regulated by AP1 factor. One of the possibility is another isoform of fos is expressed in these cells (e.g. fra1).

  1. How will you check that fra1 is expressed or not in these cells.
  2. How will you check that the heterodimer bound to the given DNA contains fra-1 along with c-jun.


My thoughts on the above questions

For first question we can do immunofluorescence assay or we can do DNA affinity assay in which we chemically synthesize DNA sequence and bind it on agarose gel column pass through it protein extract of oesophageal cancer cells and our fra-1 protein if being expressed in cell line will bind to DNA. later we can elute protein DNA complex and by anti-fra1 antibody we can check if it is being expressed. But the drawback of both these technique is we should have knowledge of epitope of fra-1.

For second question - we can perform ChIP using antibody tagged Against c-jun and then eluting out DNA bound to c-jun. Then we can simultaneously label the complex with anti fra-1 in one sample and anti fos in another sample. We can check for results. If fluorescence is obtained in case of fra-1 sample that means both c-jun and fra are bound to probe and not fos.

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  • $\begingroup$ You should add your thoughts onto the post directly and use the homework tag if it is a homework. Homework questions are on-topic as long as the OP is showing effort for answering the question himself/herself $\endgroup$ – Remi.b Mar 14 '16 at 5:27
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    $\begingroup$ I'm going to throw RT-qPCR and EMSA "supershift" out there, but there are other methods, with their own pros and cons. $\endgroup$ – CKM Mar 14 '16 at 22:09
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How will you check that fra1 is expressed or not in these cells.

RT-PCR is a quick and an easy method to check for expression. However, it only tells you about the RNA level expression and you would have to work with the assumption that RNA concentration is directly proportional to protein concentration.

Considering that you do not have an antibody for fra1, you can go for mass-spectrometric protein quantification. It is good for a high throughput analysis and is generally wasteful when you just want to study one protein. However, people do carry out targeted proteomics to study a few proteins.

Alternatively, with some effort you can actually develop an antibody for fra1.

How will you check that the heterodimer bound to the given DNA contains fra-1 along with c-jun.

Showing ternary complexes (fra-jun-DNA) are a bit difficult. It is relatively simple to show pairs. You can show that two proteins form a complex in vivo by doing a co-immunoprecipitation followed by detection by either western blot or mass-spectrometry.

You can also show interaction by biophysical techniques involving FRET and SPR but these cannot be performed in vivo (at least SPR. FRET can be done but there are limitations).

You can show the DNA-protein interaction by ChIP-seq as you mentioned in your question.

Showing all the three together is a bit tricky.

You can do it using in vitro assays that employ fluorescently labelled (one GFP and other RFP; or FRET pairs)/custom epitope tagged (for e.g. one FLAG and the other HA -tagged) proteins and a known DNA sequence generated by PCR/DNA synthesis. You can use EMSA along with antibody/fluorescence based detection to show the ternary complex.

You can indirectly infer the presence of the ternary complex when the ChIP-seq using antibodies for both the proteins shows up the same DNA sequence in the reads. You can also express a FLAG-tagged (or any other tag) protein, if in case you do not have an antibody for the proteins.

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    $\begingroup$ Nice list of many techniques, I'd just like to mention that you could also label the the DNA with biotin, an isotope or something else to suit your needs. $\endgroup$ – VonBeche Mar 11 '17 at 11:20
  • $\begingroup$ Can you clarify on the term "ternary", also ditto ^^ $\endgroup$ – hello_there_andy Mar 11 '17 at 12:00
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    $\begingroup$ @hello_there_andy "ternary" means a complex of three components. $\endgroup$ – WYSIWYG Mar 13 '17 at 17:05

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