(ChIP) assay for 3T3-L1 preadipocytes
will have to be worked out for each cell type (preads vs. ads, ST2s, etc).
Also the protocol here has not been perfected for 3T3-L1 preadipocytes yet
since I still get non-specific pulling down of DNA. More troubleshooting will
be required. But use this as a starting point…
preadipocytes to two days post-confluence in 10cm plates and treat according to
experimental design. Each plate will give around 4-6 different assays…the
exact amount will be determined after sonication.
preadipocytes once with room temp PBS.
by adding 8mL 1% Formaldehyde (in PBS) and incubating 10 min at room
3 times with cold PBS.
cells into 1mL PBS (+protease/phosphatase inhibitors) and pellet at
3000rpm for 3 min at 4C.
crude nuclei: Resuspend cells in 1mL hypotonic buffer (+inhibitors) and
incubate on ice 10 min. Dounce homogenize with 20 strokes and briefly
spin down nuclei at 13,200 rpm for 30 sec at 4C.
nuclear pellet in 250 ul 1% SDS lysis buffer (+inhibitors). Incubate on
ice 10 min.
DNA using Menon Lab cup horn sonicator (set at 4.5…see Tracy Cui in
Schwartz lab about how to use). Use 7X15 sec bursts with at least one
minute of incubation on ice between bursts. After sonication, spin down
cellular debri by centrifuging at 13,200 rpm for 5 min at 4C.
sonication, run 20ul of sample in 1.5% DNA agarose gel to verify that
sonication has resulted in 200-1000bp fragments of DNA.
protein in supernatant by standard protein assay (biorad reagent) and use
100ug protein per IP. Bring volume of each IP up to 100ul with 1% SDS
lysis buffer. Dilute 1:10 by adding 900ul of ChIP dilution buffer.
10ul for 1% input (store at –20 until time to process).
reduce nonspecific background, pre-clear each IP sample with 60ul salmon
sperm DNA/BSA/protein A agarose beads (homemade or from upstate
biotechnology), 10ul of rabbit pre-immune serum (Pierce cat# 31884) and
5ug sheared herring sperm DNA. Incubate for 1-2 hours rotating at 4C.
Pellet agarose by brief centrifugation at 1000rpm for 1 min at 4C and keep
the supernatant, discard the pellet.
the immunoprecipitating antibody (amount will vary per antibody…1-2ug) to
the supernatant fraction and incubate overnight at 4C with rotation. For
a negative control, use a non-specific antibody for immunoprecipitation.
40ul DNA/BSA/Protein A Agarose and 10ug sheared herring sperm DNA to
samples and rotate another 1-2 hours.
agarose by gentle centrifugation (700 rpm for 1 min at 4C). Carefully
remove and discard the supernatant the contains unbound, non-specific DNA.
Washt he protein A agarose/antibody/protein complex for 3-5 minutes
rotating at 4C with each of the buffers listed in the order as given
below. After final TE wash, carefully remove all liquid using a gel
loading pipette tip.
Low Salt Immune
Complex Wash Buffer (1 X 1mL)
High Salt Immune
Complex Wash Buffer (1 X 1mL)
LiCl Immune Complex
Wash Buffer (1 X 1mL)
1X TE (2 X 1mL)
prepare elution buffer (1% SDS, 0.1M NaHCO3). Recipe to make 10mL: (84mg
NaHCO3+1mL10% SDS+9mL H2O).
from beads by adding 110ul elution buffer, vortex and rotate at room temp
for 15 minutes. Carefully transfer supernatant to new tube (make sure no
beads carry-over!). Add another 110ul elution buffer to the beads and
repeat. Combine elutions into one tube so that total volume is around
10ul of 5M NaCl and 1ul Rnase (10mg/ml) to the elutions and reverse
protein/histone-DNA crosslinking by heating at 65C for 4 hours (or
overnight). At this step the sample can be stored at –20C until
next step. Remember the 1% inputs that you’ve stored at -20? Add 200ul
elution buffer, 10ul 5M NaCl and 1ul Rnase (10mg/ml) to each 10ul input
and incubate at 65C for 4 hours (or overnight) along with ChIP samples.
proteins in sample by adding 10ul of 0.5M EDTA, 20ul 1M Tris-HCl (pH 6.5)
and 2ul 10mg/ml Proteinase K for one hour at 45C.
DNA using Qiagen PCR purification kit. Final elution is in 50 ul EB.
Store sample at –20C.
PCR analysis (try 1 to 2 ul of sample for each PCR in a 25ul volume
Note: To verify
effective sonication (200-1000bp), run out 20ul of sonicated lysate in 1.5%
gel. For more accuracy (especially when troubleshooting sonication
conditions), reverse crosslinks by adding 5ul 5M NaCl to 100ul of the lysate
and incubating at 65C for 4 hours (or overnight). Then run out on gel to
verify length of fragments. Expect to see a smear of DNA from 200-1000bp (not
Note: To verify
that the immunoprecipitation of protein is working, prepare ChIP samples (with
or without IP antibody) and after step 14 (washing of beads) add 10ul Western
lysis buffer and 10ul 4X SDS LB. Heat to 95C for 10 minutes to reverse all
crosslinking and perform a western.
Recipes: no DTT in any of the recipes!!!!!!
Hypotonic Lysis Buffer: 20mM Tris-HCl, pH 7.5, 10mM NaCl, 3mM MgCl2
SDS Lysis Buffer: 1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1. (Store at RT)
Buffer: 0.01% SDS, 1.1% Triton X-100,1.2mM EDTA, 16.7mM Tris-HCl,
pH 8.1, 167mM NaCl
Low Salt Immune Complex Wash Buffer: 0.1% SDS, 1%Triton X-100, 2mM
EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl
High Salt Immune Complex Wash Buffer: 0.1% SDS, 1%Triton X-100, 2mM
EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl
LiCl Immune Complex Wash Buffer: 0.25M LiCl,1% NP40, 1%
deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 8.1.
Supplemental Protocol: To make
herring (or salmon) sperm DNA/protein A agarose
(Courtesy of Upstate
Biotechnology)…keep at 4C
1. Make 100 ml
of sterile TE (10 mM Tris, pH 8/1 mM EDTA, pH 8).
2. Combine: 50
mg BSA (the one used for diluting antibodies)
ml Sodium Azide (from a 5% stock solution)
up to 47.5 ml with TE and filter sterize using a 0.2 Ķ
3. Wash 20 ml of
protein A beads (50% slurry catalog 16-125, Upstate Biotechnologies)
using 15 ml of sterile TE.
4. Combine: 10
ml washed protein A packed beads
mg herring (or salmon) sperm DNA (sonicated)
up to 20 ml using sterile TE/BSA/sodium azide
rock 45 minutes at 4oC.
Aliquot and store at 4oC.