Name: isolation of whole cell protein lysates from mouse facial processes and cultured palatal mesenchyme cells for phosphoprotein analysis
Uploaded: 2022-04-01T15:00:00.000+00:00
Duration: 7 min 26 s
Description: Watch this Scientific Journal Video about Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis at JoVE.com

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.

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 &#176;C were used for embryo harvests. A schematic workflow of the protocol is represented in Figure 1. See the Table of Materials for details regarding all materials, equipment, software, reagents, and animals used in this protocol.
1. Harvesting E11.5 mouse embryos
<ol>
	<li>Euthanize pregnant female mouse 11.5 days after detection of vaginal plug during timed mating in a CO2 chamber using IACUC-approved CO2 flow rate required for approximately 50% displacement (2.95 L/min in a 360 in3 chamber) and duration of exposure (see Table of Materials). Perform cervical dislocation as a secondary method of euthanasia. Proceed immediately to dissection.</li>
	<li>Lay the mouse body on a dissecting board with the ventral side facing up. Spray the mouse abdomen with 70% ethanol.</li>
	<li>Open the abdominal cavity by pinching and lifting the skin anterior to the vaginal opening with straight Semken forceps and cutting the lifted skin and underlying layers with straight blade surgical scissors at a 45&#176; angle on either side to generate a &#34;V&#34; shape that extends to each lateral surface roughly half-way between the forelimbs and hindlimbs.</li>
	<li>Using the Semken forceps, grip one of the uterine horns and cut below the oviduct and above the cervix with the surgical scissors. Cut away the mesometrium to allow for complete removal of the uterine horn.</li>
	<li>Transfer the dissected uterine horn to 10 mL of histology phosphate-buffered saline (PBS) [0.137 M NaCl, 2.68 mM KCl, 1.76 mM KH2PO4 monobasic, 10.14 mM Na2HPO4 dibasic, pH 7.4] in a 10 cm Petri dish.</li>
	<li>Remove the second uterine horn on the opposing side of the abdominal cavity using the same procedure described in steps 1.4-1.5.</li>
	<li>Place the 10 cm Petri dish containing both uterine horns on ice if not proceeding immediately to the dissection of individual embryos (i.e., if a second female mouse will be dissected).</li>
	<li>Under a dissecting stereo microscope, carefully dissect out each embryo from the uterine horns with Dumont #5 fine forceps. Slowly pull away the myometrium, decidua, and chorion. Tear and remove the relatively transparent amnion surrounding the embryo, and sever the umbilical cord connecting the embryo to the placenta.</li>
	<li>Transfer each dissected embryo to 2.5 mL of histology PBS in an individual well of a 12-well cell culture plate on ice using a cut plastic transfer pipet or an embryo spoon. 
	&#8203;NOTE: If working with a litter that may contain embryos of more than one genotype, save the amnion surrounding each embryo for genotyping by placing each in its own prelabeled 0.5 mL microcentrifuge tube on ice.</li>
</ol>
2. Dissecting maxillary processes from E11.5 mouse embryos
<ol>
	<li>Prepare three 10 cm Petri dishes containing 10 mL of histology PBS and keep them on ice to be used in rotation between embryos.</li>
	<li>Transfer one embryo from an individual well of the 12-well cell culture plate to one of the 10 cm Petri dishes with histology PBS on ice using a cut plastic transfer pipet or an embryo spoon.</li>
	<li>Under the dissecting microscope, separate each maxillary process from the face using the fine forceps. First, cut the anterior side of one maxillary process along the natural indentation separating the lateral nasal process and the maxillary process (Figure 2A).</li>
	<li>Second, cut the posterior side of the maxillary process along the natural indentation separating the maxillary process and the mandibular process (Figure 2A).</li>
	<li>Third, to completely separate the maxillary process, make a vertical cut from the anterior to posterior sides of the maxillary process on the eye side of the maxillary process where the natural indentations referenced above end (Figure 2A-C).</li>
	<li>Repeat these three cuts for the maxillary process on the opposing side of the face.</li>
	<li>Using a 9&#34; Pasteur pipet with a 2 mL small latex bulb, transfer the dissected pair of maxillary processes and a small droplet of approximately 30 &#181;L of histology PBS to a labeled 35 mm Petri dish on ice (Figure 2D).</li>
	<li>Follow steps 2.3-2.7 for each embryo, rotating the three 10 cm Petri dishes containing histology PBS on ice between embryos. Place individual pairs of dissected maxillary processes in separate 35 mm Petri dishes on ice. 
	NOTE: Separation of the maxillary process ectoderm and mesenchyme (steps 2.9-2.17) can be performed after dissecting the maxillary processes of all embryos in the litter. If intact maxillary processes containing both the ectoderm and mesenchyme are desired, proceed to step 2.18 below.</li>
	<li>Prepare 250 &#181;L of fresh 2% trypsin in tissue culture PBS and 250 &#181;L of 10% fetal bovine serum (FBS) in tissue culture PBS and store on ice.</li>
	<li>Place a second, small droplet of approximately 30 &#181;L of 2% trypsin in the 35 mm Petri dish separate from the first, small droplet of histology PBS containing the maxillary processes. Transfer the pair of maxillary processes to the small droplet of 2% trypsin using the Pasteur pipet (Figure 2D).</li>
	<li>Incubate the dish on ice for 15 min.</li>
	<li>Remove the 35 mm Petri dish from the ice and place under the dissecting microscope.</li>
	<li>Using the fine forceps, slowly and carefully pull off the layer of ectoderm from each maxillary process (Figure 2E). 
	NOTE: If the ectoderm is not separating from the mesenchyme in an intact sheet, incubate in 2% trypsin at room temperature (RT) for up to 5 additional min before continuing with separation. If the tissue starts to disintegrate in the 2% trypsin, move the maxillary processes to the 10% FBS as described below to neutralize the trypsin and finish the separation of the ectoderm and mesenchyme.</li>
	<li>Once the maxillary process ectoderm and mesenchyme are separated (Figure 2F), transfer the desired tissues from the pair of maxillary processes to a third, small droplet of approximately 30 &#181;L of 10% FBS - separate from the two previous small droplets - using the Pasteur pipet (Figure 2D).</li>
	<li>Place the 35 mm Petri dish on ice to stop the trypsinization.</li>
	<li>Incubate the maxillary process tissues on ice in 10% FBS for 1-2 min, and then transfer the tissues from both maxillary processes to a fourth, small droplet of approximately 30 &#181;L of prechilled histology PBS on ice - separate from the three previous small droplets - using the Pasteur pipet (Figure 2D).</li>
	<li>Incubate the maxillary process tissues in histology PBS for 1 min while gently swirling the histology PBS around the maxillary process tissues with the tip of the Pasteur pipet to ensure that all FBS is rinsed off the tissue.</li>
	<li>Transfer the pair of maxillary process tissues to a labeled 1.5 mL microcentrifuge tube on ice using the Pasteur pipet, minimizing the transfer of histology PBS. Remove any excess histology PBS in the 1.5 mL microcentrifuge tube with the Pasteur pipet.</li>
	<li>Follow the above steps to dissect the maxillary processes from each embryo in the litter.</li>
	<li>Process the samples immediately to isolate whole cell protein lysates (below) or store at -80 &#176;C long-term. Prior to long-term storage, snap-freeze the 1.5 mL microcentrifuge tube in a bath of 100% EtOH on dry ice for 5 min.</li>
</ol>
3. Isolating whole cell protein lysates from mouse maxillary processes
<ol>
	<li>If maxillary processes were previously frozen at -80 &#176;C, thaw them on ice.</li>
	<li>Add 0.1 mL of ice-cold NP-40 lysis buffer (20 mM Tris HCl pH 8, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 2 mM EDTA; stored at 4 &#176;C) with protease and phosphatase inhibitors (1x complete mini protease inhibitor cocktail [dissolved in water; stored at -20 &#176;C], 1 mM PMSF [dissolved in isopropanol; stored at 4 &#176;C], 10 mM NaF [stored at -20 &#176;C; avoid freeze/thaw], 1 mM Na3VO4 [stored at -20 &#176;C], 25 mM &#946;-glycerophosphate [stored at 4 &#176;C]) added immediately before use on ice. 
	NOTE: Cell fractionation can alternatively be performed to isolate cytoplasmic, nuclear, membrane, or mitochondrial protein fractions.</li>
	<li>Pipet up and down 10 times with a 200 &#181;L pipetman.</li>
	<li>Vortex for 10 s, and then pipet up and down 10 times with a 200 &#181;L pipetman. Avoid generating bubbles.</li>
	<li>Incubate at 4 &#176;C for 2 h while rotating end over end using a 1.5 mL/2 mL paddle with a tube revolver.</li>
	<li>Centrifuge the samples at 13,500 &#215; g for 20 min at 4 &#176;C.</li>
	<li>Collect the supernatant to a new 1.5 mL microcentrifuge tube on ice with a 200 mL pipetman.</li>
	<li>Quantify the protein concentration with the protein assay kit, using 10 &#956;L of protein lysate + 10 &#956;L of NP-40 lysis buffer for experimental samples and 3-5 dilutions of bovine serum albumin (fraction V) (BSA) in NP-40 lysis buffer at a range of 0.25-2.0 mg/mL as protein standards.</li>
	<li>Proceed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (step 5) or quickly freeze the remaining lysates on dry ice and store at -80 &#176;C long-term.</li>
</ol>
4. Isolating whole cell protein lysates from primary and/or immortalized mouse embryonic palatal mesenchyme (MEPM) cells
NOTE: Isolation and culture of primary MEPM cells from E13.5 mouse embryos and immortalized MEPM cells have been previously described32,33,34. Stimulation of cells with growth factor (steps 4.1-4.7) can be performed prior to cell lysis. If non-stimulated cells are desired, proceed to step 4.8 below.
<ol>
	<li>Aspirate the growth medium [Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 50 U/mL of penicillin, 50 &#181;g/mL of streptomycin, 2 mM L-glutamine, 10% FBS] from the MEPM cells at ~70% confluence in a 6 cm cell culture dish using a 5.75&#34; Pasteur pipet attached to a vacuum system.</li>
	<li>Wash the cells with 1 mL of tissue culture PBS.</li>
	<li>Add 3 mL of serum starvation medium [DMEM with 50 U/mL of penicillin, 50 &#181;g/mL of streptomycin, 2 mM L-glutamine, 0.1% FBS] prewarmed in a 37 &#176;C water bath.</li>
	<li>Incubate at 37 &#176;C and 5% CO2 for 23 h.</li>
	<li>Replace the serum starvation medium with 3 mL of fresh, prewarmed serum starvation medium.</li>
	<li>Incubate at 37 &#176;C and 5% CO2 for 1 h.</li>
	<li>Stimulate the cells with the growth factor of choice at an empirically-determined concentration for the desired length of time.</li>
	<li>Aspirate the medium from the MEPM cells at 80%-100% confluence using a 5.75&#34; Pasteur pipet attached to a vacuum system.</li>
	<li>Wash the cells twice with ice-cold tissue culture PBS (1 mL for a 6 cm cell culture dish); tilt the plate to the side during the last wash to ensure all the tissue culture PBS is aspirated.</li>
	<li>Lyse the cells by adding ice-cold NP-40 lysis buffer with protease and phosphatase inhibitors added immediately before use (0.1 mL for a 6 cm cell culture dish) on ice.</li>
	<li>Incubate the plate on ice for 5 min with rotation approximately every min to ensure complete coverage of the plate.</li>
	<li>Scrape the cells off the plate using a precooled cell lifter and transfer the cell suspension to a precooled 1.5 mL microcentrifuge tube on ice.</li>
	<li>Incubate the cell suspension at 4 &#176;C for 30 min while rotating end over end using a 1.5 mL/2 mL paddle with a tube revolver.</li>
	<li>Proceed with steps 3.6-3.9 described above.</li>
</ol>
5. Western blotting of whole cell protein lysates from mouse facial processes and/or MEPM cells for phosphoproteins
<ol>
	<li>Prepare whole cell protein lysate samples for SDS-PAGE.
	<ol>
		<li>Determine the amount of protein to be loaded, depending on protein abundance in the tissue/cell; 12.5 &#956;g is usually sufficient for robustly-expressed proteins in whole cell protein lysates.</li>
		<li>Add an equal volume of 2x Laemmli buffer [20% glycerol, 4% SDS, 0.004% bromophenol blue, 0.125 M Tris HCl pH 6.8] with 10% &#946;-mercaptoethanol added immediately before use. 
		CAUTION: &#946;-mercaptoethanol is skin or eye corrosive, toxic, hazardous if swallowed, and has aquatic toxicity. Handle wearing gloves, a laboratory coat, face shield, and safety glasses in a chemical fume hood. Dispose of according to Environmental Health and Safety guidelines.</li>
		<li>Mix by vortexing.</li>
		<li>Heat the samples at 100 &#176;C for 5 min in a mini dry bath.</li>
		<li>Mix by vortexing and place the samples on ice.</li>
		<li>Centrifuge at 9,400 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Place the samples briefly on ice before loading into SDS-PAGE gel, or store at -20 &#176;C long-term.</li>
	</ol>
	</li>
	<li>Perform SDS-PAGE using an electrophoresis cell with a 4%-15% precast protein gel and electrophoresis buffer35.</li>
	<li>Electrotransfer the proteins to a polyvinylidene fluoride (PVDF) membrane using transfer buffer with 0%-20% methanol (0%-10% for proteins greater than 100 kDa; 20% for proteins less than 100 kDa) added immediately before use35.</li>
	<li>Block the membrane and probe for phosphoprotein of interest.
	<ol>
		<li>Following transfer, wash the membrane in 1x tris-buffered saline (TBS) [20 mM Tris, 0.137 M NaCl, pH 7.6] for 5 min in a western blot box.</li>
		<li>Incubate the membrane in 5 mL of blocking buffer [1x TBS, 0.1% Tween 20, 5% w/v BSA; mixed well and filtered through a 25 mm syringe filter with 0.2 &#956;m pores using a 10 mL syringe with luer tip] for 1 h in a western blot box with agitation on an orbital shaker. 
		NOTE: Do not use milk as a blocking agent when characterizing phosphoproteins as it contains the phosphoprotein casein, which can cause a high, non-specific background signal.</li>
		<li>Wash the membrane three times for 5 min each in TBS-T [1x TBS, 0.1% Tween 20] in a western blot box with agitation on an orbital shaker.</li>
		<li>Incubate the membrane in 5 mL of primary antibody diluted in blocking buffer at 4 &#176;C overnight in a 50 mL conical tube using a 50 mL paddle with a tube revolver. 
		NOTE: Consult the antibody datasheet for the appropriate concentration for western blotting.</li>
		<li>Wash the membrane three times at RT for 5 min each in TBS-T in a western blot box with agitation on an orbital shaker.</li>
		<li>Incubate the membrane in 5 mL of appropriate horseradish peroxidase (HRP)-conjugated secondary antibody diluted in blocking buffer for 1 h in a western blot box with agitation on an orbital shaker.</li>
		<li>Wash the membrane three times for 5 min each in TBS-T in a western blot box with agitation on an orbital shaker.</li>
		<li>Incubate the membrane in enhanced chemiluminescence (ECL) western blotting substrate [mixing equal volumes from bottles 1 and 2; 0.5 mL of each for large (8.6 cm x 6.7 cm) membranes] for 1 min, ensuring that the entire membrane is continually exposed to the ECL western blotting substrate.</li>
		<li>Drain the membrane of excess ECL western blotting substrate and immediately develop using a chemiluminescence imager.</li>
	</ol>
	</li>
	<li>Strip the membrane and reprobe for total protein of interest.
	<ol>
		<li>Place the PVDF membrane in a transparent pouch with polyethylene lining containing 5 mL of stripping buffer [2% SDS, 62.5 mM Tris HCl&#160;pH 6.8] with 8 &#956;L/mL of &#946;-mercaptoethanol added immediately before use.</li>
		<li>Incubate at 50 &#176;C for 30 min with rocking in a hybridization oven at 11 revolutions per minute.</li>
		<li>Wash the membrane three times at RT for 5 min each in TBS-T in a western blot box with agitation on an orbital shaker.</li>
		<li>Proceed with steps 5.4.2-5.4.9 described above.</li>
	</ol>
	</li>
	<li>Quantitate western blot band densities using ImageJ software, normalizing the levels of the phosphorylated protein of interest to the levels of the total protein of interest36.</li>
</ol>

The authors have nothing to disclose.

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...

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 the formation of distinct outgrowths - the secondary palatal shelves - from the oral side of the maxillary processes at E11.5. Over time, the palatal shelves grow downward on either side of the tongue, elevate to an opposing position above the tongue, and eventually fuse at the midline to form a continuous palate that separates the nasal and oral cavities by E16.51.
These morphological changes throughout craniofacial development are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. For example, cell membrane receptors, such as subfamilies of transforming growth factor (TGF)-&#946; receptors, including bone morphogenetic protein receptors (BMPRs), and various receptor tyrosine kinase (RTK) families, are autophosphorylated upon ligand binding and activation in cranial neural crest cells2,3,4. Additionally, the G protein-coupled transmembrane receptor Smoothened becomes phosphorylated in cranial neural crest cells and craniofacial ectoderm downstream of Sonic hedgehog (SHH) ligand binding to the Patched1 receptor, resulting in Smoothened accumulation at the ciliary membrane and SHH pathway activation5. Such ligand-receptor interactions can occur through autocrine, paracrine, and/or juxtacrine signaling in craniofacial contexts. For example, BMP6 is known to signal in an autocrine manner during chondrocyte differentiation6, whereas fibroblast growth factor (FGF) 8 is expressed in the pharyngeal arch ectoderm and binds to members of the FGF family of RTKs expressed in the pharyngeal arch mesenchyme in a paracrine fashion to initiate patterning and outgrowth of the pharyngeal arches7,8,9,10. Furthermore, Notch signaling is activated in both chondrocytes and osteoblasts during craniofacial skeletal development through juxtacrine signaling when transmembrane Delta and/or Jagged ligands bind to transmembrane Notch receptors on neighboring cells, which are subsequently cleaved and phosphorylated11. However, there are other ligand and receptor pairs important for craniofacial development that have the flexibility to function in both autocrine and paracrine signaling. As an example, during murine tooth morphogenesis, platelet-derived growth factor (PDGF)-AA ligand has been demonstrated to signal in an autocrine manner to activate the RTK PDGFR&#945; in the enamel organ epithelium12. In contrast, in murine facial processes during mid-gestation, transcripts encoding the ligands PDGF-AA and PDGF-CC are expressed in the craniofacial ectoderm, while the PDGFR&#945; receptor is expressed in the underlying cranial neural crest-derived mesenchyme, resulting in paracrine signaling13,14,15,16,17. Regardless of the signaling mechanism, these receptor phosphorylation events often result in the recruitment of adaptor proteins and/or signaling molecules, which frequently become phosphorylated themselves to initiate intracellular kinase cascades such as the mitogen-activated protein kinase (MAPK) pathway18,19.
The terminal intracellular effectors of these cascades can then phosphorylate an array of substrates, such as transcription factors, RNA-binding, cytoskeletal and extracellular matrix proteins. Runx220, Hand121, Dlx3/522,23,24, Gli1-325, and Sox926 are among the transcription factors phosphorylated in the context of craniofacial development. This post-translational modification (PTM) can directly affect susceptibility to alternative PTMs, dimerization, stability, cleavage, and/or DNA-binding affinity, among other activities20,21,25,26. Additionally, the RNA-binding protein Srsf3 is phosphorylated in the context of craniofacial development, leading to its nuclear translocation27. In general, phosphorylation of RNA-binding proteins has been shown to affect their subcellular localization, protein-protein interactions, RNA binding, and/or sequence specificity28. Furthermore, phosphorylation of actomyosin can lead to cytoskeletal rearrangements throughout craniofacial development29,30, and phosphorylation of extracellular matrix proteins, such as small integrin-binding ligand N-linked glycoproteins, contributes to biomineralization during skeletal development31. Through the above and numerous other examples, it is evident that there are wide implications for protein phosphorylation during craniofacial development. Adding an additional level of regulation, protein phosphorylation is further modulated by phosphatases, which counteract kinases by removing phosphate groups.
These phosphorylation events at both the receptor and effector molecule levels are critical for the propagation of signaling pathways and ultimately result in changes in gene expression in the nucleus, driving specific cell activities, such as migration, proliferation, survival, and differentiation, which result in proper formation of the mammalian face. Given the context specificity of protein interactions with kinases and phosphatases, the resulting changes in PTMs, and their effects on cell activity, it is critical that these parameters be studied in a physiologically-relevant setting to gain complete understanding of the contribution of phosphorylation events to craniofacial development. Here, examples of two contexts in which to study phosphorylation of proteins and, thus, activation of signaling pathways 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 - both primary32 and immortalized33. At E11.5, the maxillary processes are in the process of fusing with the lateral and medial nasal processes1, thereby representing a critical timepoint during mouse craniofacial development. Further, maxillary processes and cells derived from the palatal shelves were chosen here because the latter structures are derivatives of the former, thereby providing researchers the opportunity to interrogate protein phosphorylation in vivo and in vitro in related contexts. However, this protocol is also applicable to alternative facial processes and developmental timepoints.
A critical problem in studying phosphorylated proteins is that they are easily dephosphorylated during protein isolation by abundant environmental phosphatases. To overcome this barrier, adaptations and modifications to standard laboratory methods that allow for isolation of phosphorylated proteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphorylated proteins. 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 signaling pathways active during craniofacial development.

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

isolation of whole cell protein lysates from mouse facial processes and cultured palatal mesenchyme cells for phosphoprotein analysis

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.

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...

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

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 ...

isolation-whole-cell-protein-lysates-from-mouse-facial-processes

Research

JoVE Journal

Developmental Biology

1.9K Views.  University of Colorado Anschutz Medical Campus. 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.

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

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Biochemistry

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein ...

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.
Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

Biology

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

Neuroscience

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...