Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens

Summary

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.

Abstract

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.

Introduction

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 differences^1,2. 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 tissues^6,7. 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 °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.

Protocol

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 endothelial cells. Clinical gastroenterologists perform the endoscopy following established clinical guidelines.

1. Place the biopsy on a sterile filter paper just after endoscopy.
2. Use forceps to transfer the biopsies in 1.5 mL tubes, soak them with PBS, and place it into a cryotube.
3. Keep tubes on ice until they arrive at the laboratory.
   1. Start processing of fresh biopsies immediately.
4. For frozen biopsies, flash freeze the biopsy containing tubes and store in liquid nitrogen until use.
   NOTE: It is important to keep the sample frozen until the cold supplemented buffer 1 is added.

2. Buffer Preparation

Note: Before starting, supplement the buffers as follows.

1. Add 1 mM DTT (final concentration) and protease and phosphatase inhibitor cocktail (1x final concentration) to the hypotonic buffer 1 and hypertonic buffer 2 (Table 1).
   1. Prepare 1.5 mL of buffer 1 and 150 µL of buffer 2 per sample. DTT will prevent the oxidation of the samples.
2. Prepare nuclei wash buffer by adding 1% detergent (nonyl phenoxypolyethoxylethanol) to a final concentration of 0.1% to the already supplemented buffer 1. Prepare 1 mL of nuclei wash buffer per sample.

3. Biopsy Homogenization

1. Fresh Biopsies
   1. Place the disposable pestle in the motorized mixer. Ensure that the pestle reaches the bottom of the tube.
   2. Take the biopsy out from the cryotube and place it in a 1.5 mL tube with a pipette tip.
   3. Add 200 µL of supplemented buffer 1 to the 1.5 mL tube.
   4. Homogenize the biopsy with the pestle until the tissue is completely disrupted and keep it on ice.
   5. Once all the biopsies are homogenized proceed with step 4.
      Note: Disposable pestles must be changed between biopsies.
2. Frozen Biopsies
   CAUTION: Handle liquid nitrogen carefully; contact of liquid nitrogen with the skin or eyes may cause serious freezing injury. Protect hands and eyes always when working with liquid nitrogen .
   1. Take some liquid nitrogen in a box to store the cryotubes retrieved from the nitrogen tank.
   2. Find the biopsy in the nitrogen tank and place the cryotube in the box with the liquid nitrogen. Repeat this step for all the biopsies.
   3. Transfer the biopsy from the cryotube to a 1.5 mL tube.
      NOTE: Biopsies will be attached to the frozen tube. Push the biopsy with a sterile pipette tip so it will detach from the tube.
   4. Add 200 µL of supplemented buffer 1.
   5. Homogenize the tissue using the pestle and mortar until the tissue is completely disrupted, maintaining the temperature at 4 °C throughout all the procedure.
      Note: Do not let biopsies thaw before adding the buffer.

4. Cytoplasm Isolation

1. Incubate the homogenized biopsy on ice for 10 min.
2. Add 10 µL of 1% detergent (nonyl phenoxypolyethoxylethanol) to each sample to a final concentration of 0.05%.
   NOTE: Cells will swell and burst by osmotic lysis as the mild detergent will disrupt the cell membrane.
3. Incubate on ice for 5 minutes.
4. Vortex briefly and centrifuge at 400 x g at 4 ˚C for 2 min.
5. After vortexing, remove the supernatant and transfer it to a new clean 1.5 mL tube ; this will be the cytoplasmic fraction. Do not discard the pellet.

5. Nuclear Isolation

1. Resuspend the pellet from the previous step with 200 µL of nuclei wash buffer.
2. Centrifuge the tube for 2 min at 400 x g and 4 °C.
3. Discard the supernatant and repeat the washing procedure two more times.
   NOTE: The washes will remove cytoplasmic contamination in the nuclear fraction.
4. After the third wash, resuspend the pellet in 100 µL of buffer 2.
5. Shake the sample vigorously at 4 ˚C for 30 min. Alternatively, incubate the sample on ice and vortex every 5 min.
6. After shaking, centrifuge the sample for 10 min at top speed (> 12,000 x g) and 4 °C.
7. After vortexing, transfer the supernatant into a new clean 1.5. mL tube. This will be the nuclear fraction.

6. Protein Quantification

Note: Quantify the proteins with a bicinchoninic acid (BCA) protein assay kit. The concentration of proteins in the fractions will range between 0.5 and 5 µg/µL, being lower in the nuclear fraction (see Table 2 for protein concentrations resultant from 2 mg biopsies).

1. Prepare a standard curve with the following bovine serum albumin (BSA) amounts (µg)¹²: 0-0.5-1-2-3-4-5-6.
2. Prepare the reagents needed to use 100 µL of final BCA mix per sample.
3. Pipette 2 µL of each protein fraction in a 96 well plate, add the BCA mix and follow the manufacturer's instructions for the reading.

Results

Figure 1 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 fractionated ...

Discussion

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 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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HEPES	Sigma Aldrich	H4034-1kg
KCl	Sigma Aldrich	P9333-1kg
EDTA	Sigma Aldrich	E9884-100G
NaCl	Sigma Aldrich	5588886-1kg
DTT	Sigma Aldrich	10197777001
PMSF	Thermo Scientific	36978
Proteinase and phosphatase inhibitors	Thermo Scientific	A32959
NP-40	Sigma Aldrich	CA-630	Detergent
BCA assay	Thermo Scientific	23227	Protein quantification kit
Disposable plastic pestles	Sigma Aldrich	Z359947-100EA
Dounce homogeneizer	VWR	431-0100
Microcentrifuge	Eppendorf	5415-R
Shacker	IKA	MS3 basic