Immunoprecipitation of Target Protein-DNA Complexes from Motor Neuron Lysate

Samenvatting

Source: Montanera, K. N. et al., Rhee, H. S. High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo). J. Vis. Exp. (2020)

This video demonstrates the immunoprecipitation of DNA fragments crosslinked with target protein from stem cell-derived motor neuron lysates using target protein-specific antibody-coated magnetic beads.

Protocol

NOTE: Autoclaved distilled and deionized water (ddH₂O) is recommended for making buffers and reaction master mixes.

1. Harvesting and crosslinking cells

1. After differentiating mouse embryonic stem (ES) cells into postmitotic neurons, add 11% formaldehyde to the harvested cells to a final concentration of 1% (v/v). Rock cells on a rocking platform (a rocker, shaker, or rotator) for 15 min, at room temperature (RT, 25 °C).
   NOTE: Depending on the target protein to be crosslinked, less formaldehyde crosslinking (for example, a final concentration of 0.5%) or do....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Agarose, UltraPure	Invitrogen	16500	Checking Sonication (Section 4.3.6)
Albumin, Bovine Serum (BSA), Protease Free, Heat Shock Isolation, Min. 98%	BioShop	ALB003	Blocking Solution
Antibody against Isl1	DSHB	39.3F7	Antibody incuation with beads (Section 5.6)
Bioruptor Pico	Diagenode	B01060010	Sonicating Chromatin (Section 3.3)
Centrifuge 5424 R	Eppendorf	5404000138	Sonicating Chromatin (Section 3.5), Checking Sonication (Section 4.3.1, 4.3.3 and 4.3.4)
Centrifuge 5804 R	Eppendorf	22623508	Harvest, cross-linking and freezing cells (Section 1.3), Cell lysis (Section 2.2 and 2.3), Sonicating Chromatin (Section 3.1)
Complete, Mini, EDTA-free Protease Inhibitor Cocktail	Roche	4693159001	Added to all buffers, except Proteinase K buffer and ChIP Elution buffer
Deoxycholic Acid Sodium Salt	fisher scientific	BP349	Lysis Buffer 3, High Salt Wash Buffer and LiCl Wash Buffer
EDTA, 0.5 M, Sterile Solution, pH 8.0	BioShop	EDT111	Lysis Buffer 1-3, Checking Sonication (Section 4.1), High Salt Wash Buffer, LiCl Wash Buffer and ChIP Elution Buffer
Ethyl Alcohol Anhydrous, 100%	Commercial alcohols	P006EAAN	Checking Sonication (Section 4.3.3)
Formaldehyde, 36.5-38%, contains 10-15% methanol	Sigma	F8775	Harvest, cross-linking and freezing cells (Section 1.1)
Glycerol, Reagent Grade, min 99.5%	BioShop	GLY002	Lysis Buffer 1
Glycine, Biotechnology Grade, min. 99%	BioShop	GLN001	Harvest, cross-linking and freezing cells (Section 1.2)
Glycogen, RNA Grade	Thermo Scientific	R0551	Checking Sonication (Section 4.3.3)
HEPES, 1 M Sterile-filtered Solution, pH 7.3	BioShop	HEP003	Lysis Buffer 1, High Salt Wash Buffer
Lithium Chloride (LiCl), Reagent grade	Bioshop	LIT704	LiCl Wash Buffer
Magnetic beads for ChIP (Dynabeads Protein G)	Dynabeads Protein G (magnetic beads for ChIP)	Dynabeads Protein G (magnetic beads for ChIP)	Antibody incubation with beads (Section 5)
Magnetic rack (DynaMag-2 Magnet)	Invitrogen	12321D	Used in many steps in Sections: 5
N-Lauroylsarcosine sodium salt solution, 30% aqueous solution, ≥97.0% (HPLC)	Sigma	61747	Lysis Buffer 3
Octylphenol Ethoxylate (IGEPAL CA630)	BioShop	NON999	Lysis Buffer 1 and LiCl Wash Buffer
Phenol:Chloroform:Isoamyl Alcohol, Biotechnology Grade (25:24:1)	BioShop	PHE512	Checking Sonication (Section 4.3.1)
Phosphate-Buffered Saline (PBS), 1x	Corning	21040CV	Harvest, cross-linking and freezing cells (Section 1.3) and Sonicating Chromatin (Section 3.1), Antibody incuation with beads (Section 5.1)
PowerPac Basic Power Supply	BioRad	1645050	Checking Sonication (Section 4.3.6)
Proteinase K Solution, RNA Grade	Invitrogen	25530049	Checking Sonication (Section 4.2)
Rnase A, Dnase and Protease-free	Thermo Scientific	EN0531	Checking Sonication (Section 4.3.2)
Sodium chloride (NaCl), BioReagent	Sigma	S5886	Lysis Buffer 1-3, High Salt Wash Buffer
Sodium Dodecyl Sulfate (SDS), Electrophoresis Grade	BioShop	SDS001	Checking Sonication (Section 4.1), High Salt Wash Buffer and ChIP Elution Buffer
Sonication beads and 15 mL Bioruptor Tubes	Diagenode	C01020031	Sonicating Chromatin (Section 3.1 and 3.2)
ThermoMixer F1.5	Eppendorf	5384000020	Section 4.2, 4.3.2, and 4.3.5
Trizma hydrochloride solution (Tris-HCl), BioPerformance Certified, 1 M, pH 7.4	Sigma	T2194	10 mM Tris-HCl Buffer
Trizma hydrochloride solution (Tris-HCl), BioPerformance Certified, 1 M, pH 8.0	Sigma	T2694	Lysis Buffer 2, Lysis Buffer 3, Checking Sonication (Section 4.1), LiCl Wash Buffer and ChIP Elution Buffer
VWR Mini Tube Rocker, Variable Speed	VWR	10159-752	Used in many steps in sections: 1, 2, 5, 6 and 7
2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, Triton X-100, Reagent Grade	BioShop	TRX506	Lysis Buffer 1, Sonicating Chromatin (Section 3.4) and High Salt Wash Buffer