Name: isolation of intermediate filament proteins from multiple mouse tissues to study aging-associated post-translational modifications
Uploaded: 2017-05-18T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications at JoVE.com

This work was supported by the NIH grants NIH R01 DK110355,&#160;DK093776 [N.T.S.], DK102450 [N.T.S.], and P30 DK034987 [to UNC-Chapel Hill]. The authors thank Deekshita Ramanarayanan for assistance with qPCR and western blot experiments.

The protocol is approved and performed in accordance with the Institutional Animal Care and Use Committee (IACUC) at the University of North Carolina.
1. Preparations
<ol>
	<li>Prepare Triton-X buffer (1% Triton X-100, 5 mM ethylenediaminetetraacetic acid (EDTA), bring up volume in phosphate-buffered saline (PBS), pH 7.4). To make 500 mL: stir 5 mL each of Triton X-100 and 500 mM EDTA into 490 mL of PBS, pH 7.4. Store Triton-X buffer solution at 4 &#176;C.</li>
	<li>Prepare High Salt Buffer (10 mM Tris-HCl, pH 7...

Methods that enable biochemical characterization of IF proteins can be useful to understand numerous pathophysiological phenomena in mammalian systems, since IF proteins are both markers and modulators of cellular and tissue stress29. The principle behind the current method is based on the initial procedures developed in the 1970s and 1980s to isolate, separate and reconstitute IF proteins from cells and tissues, generally employing low and high salt solutions and Triton-X100 detergent<sup class="...

IFs are a family of proteins that in humans are encoded by 73 genes and categorized into six major types: types I-IV are cytoplasmic (e.g. epithelial and hair keratins (K), myocyte desmin, neurofilaments, glial fibrillary acidic protein (GFAP), and others); type V are the nuclear lamins; and type VI are IFs in the eye lens1. In terms of their molecular organization, IF proteins have three common domains: a highly conserved coiled-coil &#34;rod&#34; domain, and globular &#34;head&#34; and &#34;tail&#34; domains. IF protein tetramers assemble to form short filament precursors, which are ultimately incorporated into mature filaments that shape dynamic cytoskeletal and nucleoskeletal structures involved in mechanical protection2, stress sensing3,4, regulation of transcription5 and growth, and other critical cellular functions1,6,7.
The functional importance of the IF system is highlighted by the existence of many human diseases caused by missense mutations in IF genes, including neuropathies, myopathies, skin fragility disorders, metabolic dysfunctions, and premature aging syndromes8. Some IF gene mutations do not cause, but predispose their carriers to disease progression, such as the simple epithelial keratins in liver disease9. The latter is due to the critical stress-protective functions of IFs in epithelia. IFs in general are among the most abundant cellular proteins under basal conditions, but are further strongly induced during various types of stress10. For example, recent studies evaluating proteome-wide changes in the nematode C. elegans demonstrated that multiple IFs are highly upregulated and prone to aggregation during organismal aging11,12. Since maintenance of a proper IF structure is essential for cellular resistance to various forms of stress10, IF aggregation may also contribute to the functional decline during aging. However, organismal-level studies examining multiple mammalian IF proteins across different tissues undergoing stress are lacking.
IFs are highly dynamic structures that adapt to meet cellular demands. Keratins, for example, undergo a biosynthesis-independent cycling between soluble (non-filamentous) and insoluble (filamentous) protein pool13. Under normal physiologic conditions approximately 5% the total K8/K18 pool can be extracted in detergent-free buffer, in comparison to approximately 20% that can be solubilized in the non-ionic detergent Nonidet P-40, which is biochemically comparable to Triton-X10014,15. During mitosis there is a notable increase in the solubility of simple-type epithelial K8 and K1814, which is less apparent in epidermal keratins but more apparent in vimentin and other type III IF proteins15,16. Solubility properties of IF proteins are tightly regulated by phosphorylation, a key post-translational modification (PTM) for filament rearrangement and solubility17,18,19,20. Most IFs undergo extensive regulation by a number of PTMs at conserved sites, resulting in functional changes17.
The purpose of this method is to introduce investigators who are new to the IF field to biochemical extraction and analytical methods for the study of IF proteins across multiple mouse tissues. Specifically, we focus on isolation of IF proteins using a high-salt extraction method and assessment of changes in PTMs via mass-spectrometry and by PTM-targeting antibodies. These methods build upon previously published procedures21 but include modifications for extracting different IF protein types to uncover common mechanism for regulation across the IF family. For example, K8 acetylation at a specific lysine residue regulates filament organization, while hyperacetylation promotes K8 insolubility and aggregate formation22. Recent global proteomic profiling studies have additionally revealed that most tissue-specific IF proteins are also targets for acetylation and that most IF acetylation sites are confined to the highly conserved rod domain. This highlights the need for methods suitable for global profiling of the IF system. We also introduce a rapid method of isolating IF proteins from multiple tissues using automated homogenization in optimized lysing matrix. The resulting preparations are suitable for downstream PTM analysis via mass spectrometry and other methods.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dynabeads Protein G</td>
			<td>ThermoFisher Scientific</td>
			<td>10009</td>
			<td>immunoprecipitation beads</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFisher Scientific</td>
			<td>10010049</td>
			<td>for buffers</td>
		</tr>
		<tr>
			<td>Purelink RNA mini kit</td>
			<td>ThermoFisher Scientific</td>
			<td>12183018</td>
			<td>RNA extraction from tissue</td>
		</tr>
		<tr>
			<td>Purelink DNAse set</td>
			<td>ThermoFisher Scientific</td>
			<td>12185010A</td>
			<td>on column DNA digestion</td>
		</tr>
		<tr>
			<td>Dynamag-2</td>
			<td>ThermoFisher Scientific</td>
			<td>12321D</td>
			<td>magnet for use with dynabeads</td>
		</tr>
		<tr>
			<td>GelCode Blue Stain Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>24592</td>
			<td>mass spectrometry-compatible gel stain</td>
		</tr>
		<tr>
			<td>Pierce ECL Western Blotting Substrate</td>
			<td>ThermoFisher Scientific</td>
			<td>32106</td>
			<td>for use in western blot</td>
		</tr>
		<tr>
			<td>High Capacity cDNA reverse transcription kit</td>
			<td>ThermoFisher Scientific</td>
			<td>4368813</td>
			<td>for use in gene expression analysis</td>
		</tr>
		<tr>
			<td>Proflex 3x32-well PCR System</td>
			<td>ThermoFisher Scientific</td>
			<td>4484073</td>
			<td>PCR system</td>
		</tr>
		<tr>
			<td>PVDF transfer membrane&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>88520</td>
			<td>for western blot</td>
		</tr>
		<tr>
			<td>Power Up SYBR master mix</td>
			<td>ThermoFisher Scientific</td>
			<td>A25778</td>
			<td>for qPCR analysis</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>ThermoFisher Scientific</td>
			<td>AM7020</td>
			<td>solution for tissue storage prior to RNA isolation</td>
		</tr>
		<tr>
			<td>Novex 4-20% Tris Glycine Gel</td>
			<td>ThermoFisher Scientific</td>
			<td>XP04205BOX</td>
			<td>Precast protein gel</td>
		</tr>
		<tr>
			<td>Anti-Keratin 8 antibody (TS1)</td>
			<td>ThermoFisher Scientific</td>
			<td>MA514428</td>
			<td>for western blot detection of K8</td>
		</tr>
		<tr>
			<td>Anti-Vimentin</td>
			<td>ThermoFisher Scientific</td>
			<td>MA511883</td>
			<td>for western blot detection of vimentin</td>
		</tr>
		<tr>
			<td>Tris Glycine Transfer Buffer (25X)</td>
			<td>ThermoFisher Scientific</td>
			<td>LC3675</td>
			<td>for wet transfer of protein gels</td>
		</tr>
		<tr>
			<td>2X SDS Sample Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>LC2676</td>
			<td>for preparing protein gel samples</td>
		</tr>
		<tr>
			<td>Tris Glycine SDS Running Buffer (10X)</td>
			<td>ThermoFisher Scientific</td>
			<td>LC26755</td>
			<td>for running protein gels</td>
		</tr>
		<tr>
			<td>Lysing beads - Matrix D</td>
			<td>MP Biomedicals</td>
			<td>116913100</td>
			<td>Lysis beads and matrix tubes for tissue disruption and RNA extraction</td>
		</tr>
		<tr>
			<td>Lysing beads - Matrix SS</td>
			<td>MP Biomedicals</td>
			<td>116921100</td>
			<td>Lysis beads and matrix tubes for tissue disruption and protein extraction</td>
		</tr>
		<tr>
			<td>NanoDrop Lite Spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-LITE</td>
			<td>measurement of protein and nucleic acid</td>
		</tr>
		<tr>
			<td>Precellys 24 homogenizer</td>
			<td>Bertin Instruments</td>
			<td>EQ03119</td>
			<td>Automated tissue homogenizer</td>
		</tr>
	</tbody>
</table>

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.

A new rapid method for high salt-based extraction of IF proteins from multiple mouse tissues using lysing matrix. 
The traditional method25,26 of isolating the bulk of the intermediate filament protein fraction from epithelial tissue was modified here to include 9 different organs and a more rapid procedure for tiss...

Watch this Scientific Journal Video about Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications at JoVE.com

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

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. ...

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