eoe sub articles

EoE Sub Articles

1. Cell Line Preparation
<ol>
	<li>Thaw approximately 1,000,000 mouse neuroblastoma spinal cord cells (NSC-34 cells) that were stored in a liquid nitrogen container. Use a 37 &#176;C water incubator until the cells are fully thawed.</li>
	<li>Add fresh, warm medium (DMEM medium containing 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% pen-strep antibiotics).</li>
	<li>Centrifuge thawed cells (SL16R) at 500 x g for 5 min, forming a cell pellet. Discard the medium and add 1 mL of new medium for the cell resuspension.</li>
	<li>Place the resuspended cells in a 15 mL flask and add an extra 5 mL of medium. Incubate the cells overnight in a sterile incubator with 5% CO2 at 37 &#176;C.</li>
	<li>In parallel, place 8 uncoated coverslips (cover glass: 12 mm, 0.13-0.17 mm thick) in a 60-mm culture dish and sterilize using 70% ethanol and exposure to 30 min of UV light until it is dry. Add medium to each dish and dispose to wash off the residual ethanol.</li>
	<li>Add trypsin buffer (2.5 g of trypsin and 0.2 g of EDTA in 1 L of Hanks&#39; Balanced Salt Solution) to the cells after they reach approximately 90% confluence (roughly 6 million cells per flask) to allow adherent cells to dissociate. 
	NOTE: NSC-34 cells are sensitive and only require 15 s of trypsin buffer incubation and 1 additional minute of incubation free of trypsin before dissociation using the medium.</li>
	<li>Plate the cells onto the 60 mm culture dishes already containing the coverslips with 3 mL of medium, approximately 350,000 per dish. Leave them in the incubator for 24 h to allow cell attachment onto the coverslips.</li>
</ol>
2. Protein Expression Validation
<ol>
	<li>Place 60 mm culture dishes containing the residual cells (not used in the above experiment) from each experimental group on ice.</li>
	<li>Dispose of the medium and wash the cells twice with PBS to dilute and wash the remaining medium.</li>
	<li>Treat the cells with 200-400 &#181;L of lysis buffer containing PBS, 0.5% Triton, and protease inhibitor cocktail tablet (PI). Scrape using a cell scraper.</li>
	<li>Collect the cells in lysis buffer in a microcentrifuge tube and homogenize with a homogenizer at 4,000 rpm for 1 min under ice.</li>
	<li>Centrifuge the homogenized cells at 2,500 x g for 10 min. Collect the supernatant, quantify the protein concentration using a Bradford assay, and store at -20 &#176;C until use.</li>
	<li>Collect between 30 and 70 &#181;g of total protein (depending on the protein) and load it into an SDS-PAGE gel to test for protein expression using an immunoblot assay.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mouse Motor Neuron-Like hybrid Cell Line (NSC-34)</td>
			<td>tebu-bio</td>
			<td>CLU140-A</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM 4.5g/L L-glucose</td>
			<td>Biological Industries</td>
			<td>01-055-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS European Grade</td>
			<td>Biological Industries</td>
			<td>04-007-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>SL 16R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004030</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Forma Series ii Water Jacketed CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>3111</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass 12mm dia. thick 0.13-0.17mm</td>
			<td>Bar Naor LTD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axiovert 100 inverted microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leptomycin B</td>
			<td>Sigma</td>
			<td>L2913</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biological Industries</td>
			<td>02-023-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer motor/control/chuck/90-degree clamp</td>
			<td>Glas-Col</td>
			<td>099C K5424CE</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroCL 17R Centrifuge, Refrigerated</td>
			<td>Thermo Scientific</td>
			<td>75002455</td>
			<td></td>
		</tr>
	</tbody>
</table>

protein extraction from neuronal cells

Source: Shani, T., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55676">Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells.</a> J. Vis. Exp. (2017)
This protocol outlines the extraction of proteins from neuronal cancer cells. The cells are washed with a buffer and lysed via enzymatic and mechanical disruption. The extracted protein-rich supernatant is collected after centrifugation.

Protein Extraction from Neuronal Cells

1. Cell Line Preparation

	Thaw approximately 1,000,000 mouse neuroblastoma spinal cord cells (NSC-34 cells) that were stored in a liquid nitrogen ...

Take a cell culture containing neuronal cancer cells attached to a coverslip and maintain it under cold conditions.
Remove the culture medium and repeatedly wash the cells with cold phosphate buffer to eliminate the residual medium.
Add a lysis buffer containing a low concentration of non-ionic detergent and protease inhibitors.
The non-ionic detergent disrupts the cell membrane to initiate lysis.
Meanwhile, the protease inhibitors block the cellular proteolytic enzymes, protecting the proteins from enzymatic degradation.
Next, use a cell scraper to dislodge the partially lysed cells from the coverslip.
Transfer the contents to a tube containing the lysis buffer.
Under cold conditions, use a homogenizer to mechanically lyse the cells, ensuring complete cell lysis and protein release.
Centrifuge the cell lysate.
Collect the supernatant into a fresh tube.
The extracted proteins from the neuronal tumor cells are now ready for further analysis.

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

179 Aufrufe. Quelle: Shani, T., et al. Assay zur Messung des nukleozytoplasmatischen Transports in Echtzeit in Motoneuronen-ähnlichen NSC-34-Zellen. J. Vis. Exp. (2017) Dieses Protokoll beschreibt die Extraktion von Proteinen aus neuronalen Krebszellen. Die Zellen werden mit einem Puffer gewaschen und durch enzymatischen und mechanischen Aufschluss lysiert. Der extrahierte proteinreiche Überstand wird nach der Zentrifugation aufgefangen. 

Serie: Proteinextraktion aus neuronalen Zellen - Konzept

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as oxaloacetate decarboxylase in mitochondria. This article presents a series of methods for the extraction and purification of FAHD1 from swine kidney and mouse liver. Covered methods are ionic exchange chromatography with fast protein liquid chromatography (FPLC), preparative and analytical gel filtration with FPLC, and proteomic approaches. After total protein extraction, ammonium sulfate precipitation and ionic exchange chromatography were explored, and FAHD1 was extracted via a sequential strategy using ionic exchange and size-exclusion chromatography. This representative approach may be adapted to other proteins of interest (expressed at significant levels) and modified for other tissues. Purified protein from tissue may support the development of high-quality antibodies, and/or potent and specific pharmacological inhibitors.

This protocol describes how to extract fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) from swine kidney and mouse liver. The listed methods may be adapted to other proteins of interest and modified for other tissues.

Extraktion und Reinigung von FAHD1-Protein aus Schweineniere und Mausleber

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as ...

Forschung

JoVE Journal

Biochemie

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

Kombinierte Nukleotid- und Protein Extraktionen in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Protein Extraction from Neuronal Cells

Transkript

Protein Extraction from Neuronal Cells