<em>In situ</em> Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Abstract

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix.Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected. Protocol I. Preparation for fractionationThis section describes the preparation of poly-L-Lysine coated microscope coverslips and the attachment of cells prior to fractionation. If required the cells can be transiently transfected with protein expression vectors either before or after attachment. 2. Coat clean coverslips with poly-L-Lysine by incubating them in the solution for at least 1 hour on a rocking platform at 22°C. 3. Wash the coated coverslips with sterile distilled water twice and follow with a single wash with 96% ethanol. 4. Air dry the coated coverslips on a piece of filter paper and keep them in a dry container for future use (once dry they can be stacked). A. Preparation of poly-L-1. Place a poly-L-Lysine coated coverslip in a well in a 6-well plate with the coated surface facing up. 2. Seed K562 cells at a density of 7x10 5 cells/well in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and PS (penicillin 100units/ml, streptomycin 100mg/ml). In the case of K562 cells transient transfection can be performed using electroporation (0.4cm electroporation cuvettes with 1x10 7 cells in 200μl of media at 250V / 975μF) before seeding the cells. 3. Incubate the cells for 24 hours at 37°C in 5% CO2. 4. Pour off the medium and wash the cells twice with ice cold phosphate buffered saline (PBS). 5. Follow with the subcellular fractionation protocol.1. Place an uncoated coverslip in a well in a 6-well plate. 2. Wash twice in PBS prewarmed at 37°C. 3. Detach the cells by digesting with trypsin (0.03% EDTA, 0.25% trypsin) ...

Mutations in EBF3 Disturb Transcriptional Profiles and Cause Intellectual Disability, Ataxia, and Facial Dysmorphism

“…24 hr after transfection of HEK293T cells with EBF3 expression constructs or pFLAG-CMV4-cassetteA (control vector), in situ subcellular fractionation was performed. 30 Cells were incubated with CSK buffer containing 0.1% Triton-X. The cytoplasmic extracts were removed, and proteins were precipitated.…”

“…30 We treated transiently transfected HEK293T cells expressing either EBF3 WT or one of the mutants (p.Asn66Asp, p.Tyr141Cys, p.Pro177Leu, p.Arg209Trp, or p.Gln305*) to extract cytoplasmic proteins and then selectively extracted non-tightly chromatin-bound proteins and free protein aggregates within the nucleus. Detection of FLAG-tagged EBF3 proteins by immunoblotting demonstrated that all mutants were present in both the cytoplasmic and nuclear fractions (Figure 3C).…”

A clickable psoralen to directly quantify DNA interstrand crosslinking and repair

“…In preparing samples for microscopy, a pre-extraction procedure using CSK buffer was adopted to facilitate the removal of cellular constituents (such as soluble proteins) while permitting the retention of nuclear chromatin on a solid slide support [41]. Given that pre-extraction is widely applied in immunofluorescence studies and that the procedure is readily compatible with the detection of 8-POP DNA lesions (Figure 5), it is entirely conceivable that the two methods can be coupled to monitor the spatiotemporal dynamics of specific DNA damage response (DDR) proteins to sites of 8-POP-DNA damage [42].…”

The Cellular DNA Helicase ChlR1 Regulates Chromatin and Nuclear Matrix Attachment of the Human Papillomavirus 16 E2 Protein and High-Copy-Number Viral Genome Establishment

“…At 48 h posttransfection, coverslips were removed and placed in 6-well plates. Cells were sequentially extracted to remove cellular fractions as previously described (43). After each step, one coverslip for each transfection was removed and fixed in 3.7% formaldehyde to allow analysis of the retained proteins by immunofluorescence staining.…”