Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Summary

The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.

Abstract

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. These morphological changes are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. Here, two examples of physiologically-relevant contexts in which to study phosphorylation of proteins during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves. To overcome the common barrier of dephosphorylation during protein isolation, adaptations and modifications to standard laboratory methods that allow for isolation of phosphoproteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphoproteins following western blotting of whole cell protein lysates. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various phosphoproteins active during craniofacial development.

Introduction

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. In the mouse, this process begins at embryonic day (E) 9.5 with the formation of the frontonasal prominence and pairs of maxillary and mandibular processes, each of which contains post-migratory cranial neural crest cells. The lateral and medial nasal processes arise from the frontonasal prominence with the appearance of the nasal pits and eventually fuse to form the nostrils. Further, the medial nasal processes and maxillary processes fuse to generate the upper lip. Concurrently, palatogenesis is initiated with ....

Protocol

All the procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado Anschutz Medical Campus and performed in compliance with institutional guidelines and regulations. Female 129S4 mice at 1.5-6 months of age and housed at a sub-thermoneutral temperature of 21-23 °C were used for embryo harvests. A schematic workflow of the protocol is represented in Figure 1. See the Table of Materials for details r.......

Discussion

The protocol described here allows researchers to probe critical phosphorylation-dependent signaling events during craniofacial development in a robust and reproducible manner. There are several critical steps in this protocol that ensure proper collection of data and analysis of results. Whether isolating phosphoproteins from mouse facial processes and/or cultured palatal mesenchyme cells, it is imperative to move quickly and efficiently while keeping all reagents and materials on ice when indicated. The low temperature.......

Materials

Name	Company	Catalog Number	Comments
Equipment
Block for mini dry bath	Research Products International Corp	400783
ChemiDoc XRS+ imaging system with Image Lab software	Bio-Rad	1708265	chemiluminescence imager
CO2 incubator, air jacket	VWR	10810-902
Dissecting board, 11 x 13 in	Fisher Scientific	09 002 12
Electrophoresis cell, 4-gel, for mini precast gels with mini trans-blot module	Bio-Rad	1658030
Hybridization oven	Fisher Scientific	UVP95003001
Microcentrifuge 5415 D with F45-24-11 rotor (Eppendorf)	Sigma Aldrich	Z604062
Mini dry bath	Research Products International Corp	400780
Orbital shaker	VWR	89032-092
pH meter	VWR	89231-662
Power supply for SDS-PAGE	Bio-Rad	1645050
Rectangular ice pan, maxi 9 L	Fisher Scientific	07-210-093
Stemi 508 stereo microscope with stand K LAB, LED ring light	Zeiss	4350649020000000	dissecting microscope
Timer	VWR	62344-641
Tube revolver	Fisher Scientific	11 676 341
Vortex mixer	Fisher Scientific	02 215 414
Water bath	VWR	89501-472
Western blot box	Fisher Scientific	NC9358182
Materials
Cell culture dishes, 6 cm	Fisher Scientific	12-565-95
Cell culture plates, 12 well	Fisher Scientific	07-200-82
Cell lifters	Fisher Scientific	08-100-240
CO2	Airgas	CD USP50
Conical tubes, polypropylene, 50 mL	Fisher Scientific	05-539-13
Dumont #5 fine forceps	Fine Science Tools	11254-20
Embryo spoon	Fine Science Tools	10370-17
Microcentrifuge tubes, 0.5 mL	VWR	89000-010
Microcentrifuge tubes, 1.5 mL	VWR	20170-038
Pasteur pipet, 5.75"	Fisher Scientific	13-678-6A
Pasteur pipet, 9"	VWR	14672-380
Petri dishes, 10 cm	Fisher Scientific	08-757-100D
Petri dishes, 35 mm	Fisher Scientific	FB0875711YZ
Pouches, transparent, polyethylene lining	Fisher Scientific	01-812-25B
PVDF membrane	Fisher Scientific	IPVH00010
Semken forceps	Fine Science Tools	11008-13
Small latex bulb, 2 mL	VWR	82024-554
Surgical scissors	Fine Science Tools	14002-12
Syringe filter, 25 mm, 0.2 μm pore size	Fisher Scientific	09-740-108
Syringe with luer tip, 10 mL	VWR	BD309604
Transfer pipet	Fisher Scientific	13-711-22
Western blot cassette opening lever	Bio-Rad	4560000
Whatmann 3MM chr chromatography paper	Fisher Scientific	05-714-5
Reagents
4-15% Precast protein gels, 10-well, 30 µL	Bio-Rad	4561083
β-glycerophosphate disodium salt hydrate	Sigma Aldrich	G5422-25G	stock concentration 1 M
β-mercaptoethanol	Sigma Aldrich	M3148-100ML
Bovine serum albumin, fraction V, heat shock tested	Fisher Scientific	BP1600-100
Bromophenol blue	Fisher Scientific	AC403140050
Complete mini protease inhibitor cocktail	Sigma Aldrich	11836153001	stock concentration 25x
DC protein assay kit II	Bio-Rad	500-0112
DMEM, high glucose	Gibco	11965092
E7, mouse monoclonal beta tubulin primary antibody, concentrate 0.1 mL	Developmental Studies Hybridoma Bank	E7	1:1,000
ECL western blotting substrate	Fisher Scientific	PI32106	low picogram range
ECL western blotting substrate	Genesee Scientific	20-302B	low femtogram range
Electrophoresis buffer, 5 L	Bio-Rad	1610772	stock concentration 10x
Ethanol, 200 proof, 1 gallon	Decon Laboratories, Inc.	2705HC	EtOH
Ethylenediaminetetraacetic acid, Di Na salt dihydr. (crystalline powd./electrophor.)	Fisher Scientific	BP120-500	EDTA
Fetal bovine serum, characterized, US origin, 500 mL	HyClone	SH30071.03
Glycerol (certified ACS)	Fisher Scientific	G33-4
HRP-conjugated secondary antibody, goat anti-mouse IgG	Jackson ImmunoResearch Laboratories	115-035-146	1:20,000
HRP-conjugated secondary antibody, goat anti-rabbit IgG	Jackson ImmunoResearch Laboratories	111-035-003	1:20,000
Hydrochloric acid solution, 6N (certified)	Fisher Scientific	SA56-500	HCl
Igepal Ca - 630 non-ionic detergent	Fisher Scientific	ICN19859650	Nonidet P-40
Isopropanol (HPLC)	Fisher Scientific	A451-1
L-glutamine	Gibco	25030081	stock concentration 200 mM
Methanol	Fisher Scientific	A454-4
p44/42 MAPK (Erk1/2) primary antibody	Cell Signaling Technology	9102S	1:1,000; anti-Erk1/2
PDGF-BB recombinant ligand, rat	Fisher Scientific	520BB050
PDGF Receptor β primary antibody	Cell Signaling Technology	3169S	1:1,000
Penicillin-Streptomycin	Gibco	15140122	stock concentration 100 U/mL, 100 µg/mL
Phenylmethanesulfonyl fluoride, 99%	Fisher Scientific	AC215740100	PMSF; stock concentration 100 mM
Phospho-p44/42 MAPK (Erk1/2) primary antibody	Cell Signaling Technology	9101S	1:1,000, anti-phospho-Erk1/2
Phospho-PDGF Receptor α /PDGF Receptor β primary antibody	Cell Signaling Technology	3170S	1:1,000
Potassium chloride (white crystals)	Fisher Scientific	BP366-500	KCl
Potassium phosphate monobasic (white crystals)	Fisher Scientific	BP362-500	KH2PO4
SDS solution, 10%	Bio-Rad	161-0416
Sodium chloride (crystalline/biological,certified)	Fisher Scientific	S671-3	NaCl
Sodium fluoride (powder/certified ACS)	Fisher Scientific	S299-100	NaF; aliquot for one time use; stock concentration 1 M
Sodium orthovanadate, 99%	Fisher Scientific	AC205330500	Na3VO4; stock concentration 100 mM
Sodium phosphate dibasic anhydrous (granular or powder/certified ACS)	Fisher Scientific	S374-500	Na2HPO4
Tissue culture PBS	Fisher Scientific	21-031-CV
Transfer buffer, 5 L	Bio-Rad	1610771	stock concentration 10x
Tris base (white crystals or crystalline powder/molecular biology)	Fisher Scientific	BP152-1
Trypsin	BioWorld	21560033
Tween 20	Fisher Scientific	BP337-500
Western blot molecular weight marker	Bio-Rad	1610374
Software
ImageJ software	National Institutes of Health
Animals
Female 129S4 mice			gift of Dr. Philippe Soriano, Icahn School of Medicine at Mount Sinai