The modified histone peptide array is used to screen antibodies, enzymes and proteins for crossreactivity or binding interactions

with histones and their post translational modifications. This array

screens 59 acetylation, methylation, phosphorylation and citrullination modifications on the N-terminal
tails of histones H2A, H2B, H3 and H4.
It works much like a western blot, but instead of a membrane samples are placed on the array, then
followed by blocking, washing, probing and detection via colorimetric or ECL methods.
In a pull down assay, it is the goal to understand which proteins interact with each other, as this helps to
elucidate gene function. Using an immobilized bait and prey system, it is possible to elute out proteins
that interact, and can be viewed via PAGE or western blots. Here, biotinylated H3 peptides were used to
pull down proteins coded by the genes of interest such as Pygo2, and analyzed via WB.
RNA immunoprecipitation works much in the same way as ChIP, but instead of the chromatin, it
investigates binding of proteins to RNA. By lysing cells, fixing the interactions, using an antibody against
the protein of interest to separate the RNA-protein complex and extracting the separated RNA, one can
use RT-qPCR to analyze binding levels.
Lentiviruses are used as vectors for gene delivery. What makes them unique is their ability to integrate
into the genome of non-dividing cells, unlike other retroviruses which can only infect dividing cells. It
genome is in the form of RNA, which is reverse transcribed and randomly integrated into the host
genome via the integrase enzyme. Site of integration is unpredicatable, however it has been observed
that lentiviruses have a lesser tendency to insert their genome into areas that can cause cancer as
compared to other retroviruses.
In order to prepare the virus for transduction, several plasmids are transfected into the HEK293 cell line,
one or more plasmids coding for the viral proteins such as the capsid and the reverse transcriptase,
while another plasmid codes for the genetic material to be delivered by the virus. It must be noted that
lentiviral vectors are packaged without the gene required for their replication, in order to control the
environment in which it is placed. The packaged virus is then used to infect the cells of interest and
deliver the gene. In this experiment, the lentivirus was used to transduce a cell line that expresses a
doxycycline inducible shRNA against the gene targets of interest.
Cell proliferation is also monitored during the course of the experiment, and uses the CellTiter 96
AQueous One Solution Cell Proliferation Assay to do so. In this assay, a tetrazolium compound is added,
and cell viability is measured via the quantification of a colored formazan product, which is a reduction
product that comes about due to NADPH of viable cells.
Chromosome conformation capture, as the name suggests, is used to analyze chromosome organization.
DNA and other biomolecules in the cell are first cross linked, then digested with enzymes to separate
non cross linked DNA from chromatin. Intramolecular ligation of relevant proximal DNA fragments then
occur, then the cross links are reversed. Analysis and quantification can then be done via qPCR, or ChIP.
Here it is used to determine and to confirm enhancer-promoter interactions.