Question

You have a transcription factor and you are interested to know which promotors does it bind to. You will perform the series of experiments below, but they are out of order. Put them in order.

1.) Incubate with beads that have an antibody that binds to the transcription factor of interest

2.) Shear Chromatin into fragments

3.)solate chromatin

4.)Treat cells with formaldehyde

5.)Immunoprecipitate chromatin fragments

6.)Reverse the crosslinks

Answer 1

Ans- Correct order is -

1. Treat cells with formaldehyde. ( Formaldehyde fixes cellular structure by cross- linking the proteins. DNA and associated proteins on chromatin in cells are cross linked).

2. Isolate chromatin.

3. Shear chromatin into fragments.( Chromatin fragmentation can be done by sonication, nuclease and restriction digestion process).

4. Immunoprecipitate chromatin fragments. ( Immunoprecipitation is the process of precipitating a particular protein out a solution using an antibody that specifically binds to that particular protein).

5. Reverse the crosslinks.

6. Incubate with beads that have an antibody that bind to transcription factor of interest.