Welcome to the Novus Visual Protocol Series.
In this video
you will learn how to perform all phases of chromatin immunoprecipitation
from lysate prep to DNA quantification.
This visual ChIP protocol follows the written version located on the support
section of our website
as well as the protocol for the Novus ChromataChIP Kit.
This kit includes all the components necessary for you to perform a
successful ChIP the first time you use it.
ChIP can be effectively completed with either cell culture or tissue lysate.
We will demonstrate this procedure with one flask of 80% confluent
HeLa cells.
Start by adding formaldehyde directly to the medium
to reach a final concentration of 1%.
Incubate at room temperature for 10 minutes.
To quench the crosslinking reaction,
we next add enough glycine to the flask to reach a concentration of 125
mM, or 1x if you have a 10x solution.
Incubate for five minutes at room temperature.
Remove all medium and rinse twice with ice cold PBS.
Next
add four mills of ice cold PBS
that also contains appropriate protease inhibitors
to prevent degradation of your proteins.
Scrape the cells and solution
and transfer to a 15 mill conical tube.
Pellet the cells by centrifugation at 4 degrees Celsius for 5 minutes at
800g.
Discard the supernatant and re-suspend the cells in 400 microliters of
RIPA lysis buffer and appropriate protease inhibitor.
Transfer the cells to a siliconized microcentrifuge tube and incubate on ice for
fifteen minutes.
After this you are ready for DNA shearing.
In the second phase of ChIP
we will use a sonicator equipped with a microtip to sheer our DNA to fragment
sizes of 100 to 1000 base pairs.
The conditions for sheering must be empirically derived and optimized before
proceeding to any of the following phases.
Results vary depending on your equipment,
cell type,
cell density,
cross linking efficiency and sheer conditions.
However, there are several general tips for successful DNA sheering.
First keep the body of the sonicator tip close to the bottom of the tube
and limit the volume to 400 microliters.
This will prevent foaming of the sample, as foaming decreases the efficiency of
the sonification procedure.
In addition,
perform a time course of multiple short course sonications instead of fewer
longer treatments.
This minimizes heat buildup
and unwanted DNA train of your chromosome.
For example,
we find that six, fifteen second pulses at 50% power output
worked well for us.
Placing the sample back on ice to rest for at least 60 seconds between
pulses
will cool the sample back down.
After the samples have been successfully sheered,
centrifuge each at 4 degrees Celsius
for 5 minutes at 12500g.
Remove the supernatant and transfer to a new tube
or aliquot into 10,
40 microliter samples.
These samples can safely be stored at -80 degrees Celsius
for up to one month.
To test for sheering efficiency while optimizing for this phase,
and optionally afterwards,
run 1 aliquot on 1.5% agarose gel.
To do this,
dilute the 40 microliter sample with 160 microliters
of IP dilution buffer and eight microliters of five molar sodium
chloride.
All buffers are detailed in the written protocols found in the support section
of the Novus website.
Incubate at 95 degrees Celsius for 15 minutes.
Finally,
run 10 microliters of the diluted sample with loading buffer and DNA
binding dye alongside a 100 base the perladderate to verify fragment size.
Here is an example of DNA that has been under sheered in lane two,
perfectly sheered in lane three
and over sheered in lane four.
And here is a spectral image of the same gel.
Note the even distribution of fragment sizes in lane three.
You can now proceed to the next precipitation phase.
In the third phase of ChIP
we will use a Novus Antibody
that is specific for our protein of interest
to pull down DNA that is cross linked to that protein.
This is done by using magnetic beads conjugated to a protein A/G
that will bind to the antibody protein
DNA complex.
Start by diluting samples 1 to 10
by adding 40 microliters of sheered chromatin
to 360 microliters
of the IP dilution buffer
and include protease inhibitors.
All buffers are detailed in written protocols found in the support section
of the Novus website.
It is beneficial to IP at least three samples.
One will be for your antibody of interest,
another will be for a positive control antibody
and a third for a negative control which will not contain any antibody
and will be used to measure background levels.
If you are using
the ChromataChIP Kit, your kit includes a positive control antibody.
Dilute one last sample and label this input.
This sample will not go through the IP process and instead be saved for Phase Four
of the protocol.
Store this at
four degrees Celsius until ready for use.
Add your antibody of interest and positive control antibody
to the respective diluted samples
then incubate overnight at four degrees Celsius with rotation.
The next day,
for each sample to be IPed
you will use 25 microliters of micro
fully re-suspended protein A/G magnetic beads.
First wash the beads by adding
one mL of IP dilution buffer
to the amount of beads that you will be using for all of your samples
and then vortex.
Then pellet the beads with a magnetic separator
and re-suspend the beads with the same volume of IP dilution buffer as
your original volume of beads.
Add 25 microliters of wash beads to each sample
and incubate tubes with rotation
for one hour at 4 degrees Celsius.
Next we can begin our wash steps.
Wash steps can be performed manually through the use of
the rotator and magnetic separation rack
or automated with a machine designed for this use.
Start by pelleting the beads and removing the supernatant.
Then add 500 microliters of cold IP wash buffer one
and wash for 5 minutes at 5 degrees Celsius with rotation
or agitation.
Repeat this entire step for IP wash buffers 2, 3, and 4.
Elute the complex from the beads by adding 200 microliters of
IP elution buffer and washing for 15 minutes at 4 degrees
Celsius
with rotation or agitation.
Pellet the beads
keeping the supernatant
and discarding the beads.
In phase four,
we will reverse our initial cross-linking proteins to DNA,
followed by purification of the DNA.
Start by taking each IP sample
as well as the input sample which did not go through the preceding IP
steps
and add 8 microliters of five molar sodium chloride
to the two hundred microliter samples
followed by vortexing.
Incubate at 95 degrees Celsius
for 15 minutes.
To purify each sample
we will use a DNA binding silica cone
according to the manufacturer's instructions.
First adding each sample for the proper amounts of DNA binding buffer
in vortex,
then transfer each sample into a column placed inside a collection tube.
Centrifuge the columns at 15,000g for 1 minute.
Discard the flow through from the collection tube
and add DNA wash buffer to each column.
Centrifuge the columns at 15000g for one minute.
tube and spin the empty columns at 15000g for two minutes
to ensure sure that they are completely dry.
Discard the old collection tube and place the purification column into a new clean
tube.
Add 50 microliters
of DNA elusion buffer
directly to the membrane in the column.
Allow to sit for one minute
then perform a final spin at 15000g
for one minute.
Discard the purification column
and save the purified DNA
at -20 degrees Celsius
until ready for the last phase.
In this final phase of ChIP
our precipitated and purified DNA can now be measured
through a variety of techniques such as traditional pcr
through a variety of techniques such as traditional PCR,
quantitative real time PCR, Microarray and ChIP sequencing.
Here, we will use QRTCPR to quantitate the amount of protein
associated with the specific gene locus
with the use with the use of specific primer sets.
Multiple loci can be analyzed by using different primer sets.
Start by making a master mix for each primer set that will be used,
adjusting volume for your amount samples.
If using the ChromataChIP kit, your kit includes a positive control of human
primer set.
Two microliters of purified DNA sample template can then be added directly to
the wells containing your master mix.
For the input sample, first dilute the sample down to 1% of the
original concentration before adding into the wells.
Using the input sample of full concentration,
will typically result in varied data.
After performing the RTCPR, according to the manufacturer's instructions, for both
the thermocycler
and DNA polymerase mix that you use, the data can now be normalized.
The final data is typically represented
as a percentage of amplified sample signals
relative to the input sample
which contains the maximum signal possible.
More detailed instructions on how to normalize your data can be found in the
support section on the Novus website (www.novusbio.com).