Name: subcellular fractionation from fresh and frozen gastrointestinal specimens
Uploaded: 2018-07-15T15:00:00
Description: Watch this Scientific Journal Video about Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens at JoVE.com

Authors acknowledge the assistance of Ander Lopez and Miren Telletxea for video recording and editing. ACR is funded by an Ikerbasque fellowship and a research project from Asociaci&#243;n de Celiacos Madrid (ACM). JRB is funded by Project ISCIII-PI16/00258 and co-funded by the European Union ERDF/ESF "A way to make Europe". IS is funded by a research project grant 2015111068 of the Basque Department of Health. IRG and AJM are supported by predoctoral fellowship grants from the UPV/EHU and the Basque Department of Education, respectively.

This study was approved by the Cruces University Hospital Ethics Board and analyses were performed after informed consent was obtained from all subjects or their parents.
1. Biopsy Collection
NOTE: Biopsy specimens from the distal duodenum of patients are obtained during routine diagnosis endoscopy managing biopsies that range between 2 and 10 mg in weight. Biopsied tissue is a complex tissue composed of different cell types including epithelial, immune and endothelia...

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The protocol described here is used for the nuclear and cytoplasmic fractionation of intestinal biopsies. The purified proteins are not denatured and can be used not only in western blot analysis as shown in Figure&#160;2 and 3 but also in assays requiring native-folded proteins such as immunoprecipitation, electrophoretic mobility shift assay (EMSA) or native polyacrylamide gel electrophoresis (PAGE).
The described method relies on mechanical hom...

Proteins participate in almost all biological functions within a cell and any variation in their structure, quantity or location can lead to a pathogenic scenario. Tissue sample fractionation methods are a useful approach to reduce the complexity of disease related protein analyses. Some studies use protein localization information or enrich a protein from a specific cellular compartment, so a protocol for reproducible fractionation of intact proteins is useful to answer certain biological questions. Determining the subcellular localization of proteins and monitoring their compartmental redistribution or interactions at basal and disease conditions will help identify disease related functional differences1,2. Thus, this method aims to reproducibly fractionate intestinal tissue biopsies, both fresh and frozen, into cytoplasmic and nuclear subcellular compartments.
Intestinal biopsy samples are routinely obtained during endoscopy procedures3 (Figure 1) and can be effectively used for protein quantification or immunoprecipitation studies. Since tissue samples from intestinal epithelial will contain proteins that may be directly involved in the disease pathogenesis4, intestinal biopsy samples are a very valuable source for the successful identification of intestinal disease-specific proteins. Stored frozen, patient tissue samples together with the clinical information are useful resources for protein analysis, and a simple and reproducible sample preparation is a key issue to provide cell compartmental information using limiting amounts of frozen tissue5.
There are several commercially available kits for the separation of cellular fractions, but these are more expensive and generally more time consuming than the protocol presented here. Protocols based on multiple-step gradient centrifugation and continuous gradients have also been previously used for the fractionation of diverse cellular compartments in different tissues6,7. However, maintaining the consistency of continuous gradients is often a difficult task. Similar protocols based on the sequential lysis of membranes have been previously described but they generally need more than two buffers and the hands on time is longer8 .
When fractionating tissue samples, bear in mind that tissue samples present cell-cell and cell-matrix interactions that are both not present in cultured cells and important when proceeding to protein extraction. The proper breakdown between cells and extracellular matrix without affecting the protein quality will be a critical factor in the intestinal tissue protein isolation9. In this protocol, cell-cell and cell-matrix contacts are first broken, releasing the cells and allowing the buffer to reach the individual cells. To avoid protein-compartment mixing prior to fractionation, the breakdown of the contacts must occur without altering the integrity of the cells or the nuclear membranes.
Due to the enrichment of various proteases in the intestinal mucosa10, it is important to control the protein degradation during extraction. To minimize the potential protein degradation, multiple protease inhibitor must be included in the protein extraction buffers. Additionally, if extracts are going to be used for functional assays, it is essential to avoid the denaturation of proteins or proteolysis as this will cause a loss of protein activity11. For this purpose, the protocol is performed at 4 &#176;C and fresh phenylmethylsulfonyl fluoride (PMSF) is added to the buffers at a final concentration of 0.1 mM just prior to use to further inhibit proteolysis.
The first step in cell fractionation is tissue disruption and cell lysis. As mentioned previously, the objective is to disaggregate the cells and break them open with minimum damage. Intestinal tissue samples must be homogenized, and the cells lysed to achieve maximum breakage of the cell membrane. In this protocol, we use a motorized pestle mixer to break the intestinal tissue which is homogenized within seconds by means of high speed vortexing action. After homogenization, tissue cells are incubated in a hypotonic buffer that will burst the cell membrane but will keep the nuclei intact, followed by addition of a non-denaturing detergent (nonyl phenoxypolyethoxylethanol) and a short vortex to separate nuclei from the cytoplasmic fraction. After the removal of the cytoplasm, the cell nuclei membrane is burst into a hypertonic buffer with shaking.
The method presented here is an appropriate method for the subcellular fractionation of fresh and frozen intestinal biopsies that have been obtained during endoscopic procedures and range between 2 and 10 mg in weight. The protocol is easy and reproducible and could be performed using basic laboratory equipment and reagents in under an hour.

<table border="0" cellpadding="0" cellspacing="0" style="width:657px;" width="658">
	<tbody>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">HEPES</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">H4034-1kg</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">KCl</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">P9333-1kg</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">EDTA</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">E9884-100G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">NaCl</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">5588886-1kg</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">DTT</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">10197777001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">PMSF</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:119px;">36978</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Proteinase and phosphatase inhibitors</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:119px;">A32959</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">NP-40&#160;</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">CA-630</td>
			<td>Detergent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">BCA assay</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:119px;">23227</td>
			<td>Protein quantification kit</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Disposable plastic pestles</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">Z359947-100EA</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Dounce homogeneizer</td>
			<td style="width:128px;">VWR</td>
			<td style="width:119px;">431-0100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Microcentrifuge</td>
			<td style="width:128px;">Eppendorf</td>
			<td style="width:119px;">5415-R</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Shacker</td>
			<td style="width:128px;">IKA</td>
			<td style="width:119px;">MS3 basic</td>
			<td></td>
		</tr>
	</tbody>
</table>

subcellular fractionation from fresh and frozen gastrointestinal specimens

The purpose of this protocol is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the localization analysis of specific proteins or protein complexes in different tissue states (i.e., healthy vs. disease). This method is useful for the fractionation of both fresh and frozen intestinal tissue samples; it is easily accessible for all laboratories and not time consuming.

Figure 1&#160;shows a hematoxylin eosin staining of an intestinal biopsy section with intact villi and crypt structures.
Representative results of nuclear and cytoplasmic fractionation using this protocol are shown in Figures 2 and 3. In the experiment shown in Figure 2, a fresh (Fh) and a frozen (Fz) biopsy were simultaneously fraction...

Watch this Scientific Journal Video about Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens at JoVE.com

Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens

Here, we present a protocol to perform a simple cellular fractionation for the subcellular separation of cytoplasmic and nuclear proteins in human fresh and frozen intestinal biopsies.

The purpose of this protocol is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the ...

The goal of the following protocol is to generate subcellular fractions from frozen or fresh gastrointestinal specimens. Proteins take part in almost all of your biological functions within a cell. In any variation they are extracted amongst a location can lead to a pathogenic scenario.Some studies will take advantage of the assessment of protein localization or will need to invade a protein from a specific cellular compartment. So the reproducible fractionation of intact proteins is important in this respect. Intestinal biopsy samples are recently obtainable during endoscopy procedures and can be effectively used for protein quantification or immuno precipitation studies.Clinical intestinal samples are an important resource for the effective identification of tissue and disease specific proteins. The name of this method is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the localization analysis of a specific protein or protein complexes. This method serves for the fractionation of both.First, our frozen intestinal biosis happens. For the frozen tissue procedure, freeze samples immediately after extraction and store in liquid nitrogen until use. It is important to maintain the sample frozen until the cold homogenization buffer is added.For fresh samples, transfer the biopsy to a 1.5 mL microtube and keep on ice until the procedure is started. Frozen tissue samples are usually attached to the wall of the cryotube, the tissue can be released using a sterile pipette tip before transferring to a conical 1.5 mL microtube. Add 200 mL of buffer one supplemented with DDT and phosphatase and protease inhibitors to the microtube containing the sample.Homogenize the sample with a vessel until the tissue is completely disrupted and keep it on ice. Incubate the sample on ice for 10 minutes. Add 10 mL of 1 percent detergent to each sample to a final concentration of 0.05 percent in order to disrupt the cell membrane.Incubate on ice for five minutes. Vortex briefly and centrifuge at 400 g at 4 degrees for two minutes. Remove the supernatant containing the cytoplasmic protein fraction and transfer it to a clean tube.Do not discard the pellet. Re suspend the pellet in 200 mL of buffer two supplemented with DDT and phosphatase and protease inhibitors. Centrifuge at 400 g for 2 minutes.Discard the supernatant and repeat the washing procedure two more times. After the third wash, re suspend the pellet in 100 mL of buffer two. Shake vigorously at 4 degrees for 30 minutes.Centrifuge at 4 degrees at top speed for 10 minutes. Transfer the supernatant containing the nuclear protein fraction to a clean tube. Cytoplasmic and nuclear fractions are now ready for quantification and downstream experiments.The intestinal mucosa is rich in several proteases and therefore protein degradation has to be controlled during extraction in order to minimize protein degradation, multiple protease inhibitors should be included in the protein extraction buffers. Additionally, the extracts are to be used for additional assays. It is essential to avoid protein denaturation or proteolysis.As this cause a loss of protein activity. As a consequence of the cell-cell and cell-matrix contacts that are present in tissues, some modifications have to be implemented in the protein extraction protocols routinely used with cell cultures. Separation of the cells from the transcellular matrix without affecting protein quality is a critical factor for the efficient isolation of intestinal tissue proteins.To the left, the table shows the protein concentrations of each of the fractions from the fresh and frozen 2 mL biopsy samples. Concentrations are shown in microgram per microliter. In this experiment, a fresh and frozen sample were simultaneously fractionated following the protocol.And a Western blot was performed using 20 mg of each sample. To check the purity of the fractions, HSP90 and Tubulin were used as cytoplasmic controls and HDAC1 and H3 as nuclear controls. As can be observed, HSP90 and Tubulin are primarily located in the cytoplasm and HDAC1 and H3 in the nucleus with very residual mixing between the two fractions.To see whether the protocol affects cell death, we also detected CASP3. Both samples present a very minimal staining for CASP3 confirming that the procedure does not affect cell death pathway even after flash-freezing the biopsies. The frozen samples used in this experiment came from an individual with an intestinal inflammatory condition and as expected, pSTAT1 appears in the cytoplasm of this sample, confirming the protein modifications are maintained after fractionation, and thus can be used for downstream functional analysis.Reproducibility of the results in frozen biopsies is shown in this figure. Fractions are clean with minimal compartment protein mixing. Fresh and frozen biopsies can be equally used in these protocols, with frozen biopsies giving clean and reproducible results as seen in Figure 1 and 2.The protocol described here is designed to generate the possible protein fractions. However, some intrinsic variability within the sample including the size and status of the tissue can lead to deviations in the protein amounts and the distribution amounts in the fractions.

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