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 ....

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 fil....

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