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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.
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.
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 ....
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.......
When attempting to characterize the phosphorylation of proteins isolated from mouse facial processes and/or cultured palatal mesenchyme cells, the representative results will ideally reveal a distinct, reproducible band following western blotting with an anti-phosphoprotein antibody that runs at or near the height of the corresponding total protein band (Figure 3). However, if extensive phosphorylation of the protein occurs, there may be a slight upward shift of the phosphoprotein band compa.......
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.......
129S4 mice were a gift from Dr. Philippe Soriano, Icahn School of Medicine at Mount Sinai. This work was supported with funds from the National Institutes of Health (NIH)/National Institute of Dental and Craniofacial Research (NIDCR) R01 DE027689 and K02 DE028572 to K.A.F., F31 DE029976 to M.A.R. and F31 DE029364 to B.J.C.D.
....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 |
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