We thank Zehavit Frenkel and Sandra Tolchinsky for technical assistance. Research related to this work is supported by grants from the Israel Science Foundation (1229/07) and German-Israeli Project Cooperation (DIP-DFG).

The following protocol is intended for analysis of sugar chains of total glycoproteins or of a specific glycoprotein of interest. The procedure is basically the same for both cases with a few alterations as stated throughout the protocol.
<ol>
 <li> For metabolic labeling of N-linked glycans one needs to use a subconfluent culture of mammalian cells grown overnight in a 90 mm dish for each sample (samples may represent different time points or treatments). This protocol is optimized for NIH 3T3 cells. </li>
 ...

<ol>
	<li>Parodi, A. J., Behrens, N. H., Leloir, L. F., Carminatti, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+polyprenol-bound+saccharides+as+intermediates+in+glycoprotein+synthesis+in+liver.">The role of polyprenol-bound saccharides as intermediates in glycoprotein synthesis in liver.</a> Proc Natl Acad Sci U S A. 69 (11), 3268-3272 (1972).</li><li>Avezov, E., Frenkel, Z., Ehrlich, M., Herscovics, A., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum+(ER)+mannosidase+I+is+compartmentalized+and+required+for+N-glycan+trimming+to+Man5-6GlcNAc2+in+glycoprotein+ER-associated+degradation.">Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5-6GlcNAc2 in glycoprotein ER-associated degradation.</a> Mol Biol Cell. 19 (1), 216-225 (2008).</li><li>Frenkel, Z., Gregory, W., Kornfeld, S., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum-associated+degradation+of+mammalian+glycoproteins+involves+sugar+chain+trimming+to+Man6-5GlcNAc2.">Endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to Man6-5GlcNAc2.</a> J Biol Chem. 278 (36), 34119-34124 (2003).</li><li>Hosokawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+endoplasmic+reticulum+(ER)+degradation+of+misfolded+Null+Hong+Kong+alpha1-antitrypsin+by+human+ER+mannosidase+I.">Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I.</a> J Biol Chem. 278 (28), 26287-26294 (2003).</li><li>Jakob, C. A., Burda, P., Roth, J., Aebi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+misfolded+endoplasmic+reticulum+glycoproteins+in+Saccharomyces+cerevisiae+is+determined+by+a+specific+oligosaccharide+structure.">Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure.</a> J Cell Biol. 142 (5), 1223-1233 (1998).</li><li>Tolchinsky, S., Yuk, M. H., Ayalon, M., Lodish, H. F., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-bound+versus+secreted+forms+of+human+asialoglycoprotein+receptor+subunits.+Role+of+a+juxtamembrane+pentapeptide.">Membrane-bound versus secreted forms of human asialoglycoprotein receptor subunits. Role of a juxtamembrane pentapeptide.</a> J Biol Chem. 271 (24), 14496-14503 (1996).</li></ol>

The pulse-chase analysis of glycans in live cells with HPLC separation provides a method for studying the dynamics of oligosaccharide structural alterations throughout the life of a glycoprotein. There is growing evidence that such alterations are involved in producing the signals for ER folding, quality control and trafficking systems 2-5. The method can be applied not only for a specific glycoprotein of interest but also for analyzing the dynamics of the structure of total glycoproteins, as illustrated in th...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>600E Multisolvent delivery System controller</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT062710</td>
<td>&nbsp;</td>
<tr>
<td>Acetonitrile LiChrosolv (gradient grade for liquid chromatography)</td>
<td>&nbsp;</td>
<td>Merck Bioscience</td>
<td>1.0003</td>
<td>&nbsp;</td>
<tr>
<td>Microcon Amicon Ultra 0.5 ml 30K or Centricon ultracel YM-30</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC503024 or 4208</td>
<td>&nbsp;</td>
<tr>
<td>Concentrator 5301, incl. 48 x 1.5 / 2.0 ml fixed-angle rotor</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5301 000.016</td>
<td>&nbsp;</td>
<tr>
<td>Dialyzed Foetal Calf Serum</td>
<td>&nbsp;</td>
<td>Biological Industries</td>
<td>04-011-1A</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Modi&#xFB01;ed Eagle's Medium</td>
<td>&nbsp;</td>
<td>Gibco Invitrogen Cell Culture</td>
<td>41965039</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Modi&#xFB01;ed Eagle's Medium - glucose free</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D5030</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D1408</td>
<td>&nbsp;</td>
<tr>
<td>EDTA disodium salt (Titriplex Iii)</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>1084180250</td>
<td>&nbsp;</td>
<tr>
<td>Endo Hf Kit (1,000,000 units/ml)</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>p0703S</td>
<td>&nbsp;</td>
<tr>
<td>Foetal Calf Serum (FCS)</td>
<td>&nbsp;</td>
<td>Biological Industries</td>
<td>04-001-1A</td>
<td>&nbsp;</td>
<tr>
<td>Frac-100 fraction collector</td>
<td>&nbsp;</td>
<td>Amersham Biosciences</td>
<td>18-1000-77</td>
<td>&nbsp;</td>
<tr>
<td>LS 6500 Liquid Scintillation Counting systems</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>510720</td>
<td>&nbsp;</td>
<tr>
<td>Mannose, D-[2-3H(N)]- (Specific Activity: 15-30Ci/mmol)</td>
<td>&nbsp;</td>
<td>PerkinElmer Life Sciences</td>
<td>NET570A</td>
<td>&nbsp;</td>
<tr>
<td>N-carbobenzoxyl-leucinyl-leucinyl-leucinal (MG-132)</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>474790</td>
<td>&nbsp;</td>
<tr>
<td>N-glycosidase F (1000 units/ml)</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11365177</td>
<td>&nbsp;</td>
<tr>
<td>N-octylglucoside</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>O3757</td>
<td>&nbsp;</td>
<tr>
<td>NIH 3T3 cells</td>
<td>&nbsp;</td>
<td>American Type Culture Collection (ATCC)</td>
<td>CRL-1658</td>
<td>&nbsp;</td>
<tr>
<td>Opti-Flour</td>
<td>&nbsp;</td>
<td>PerkinElmer Life Sciences</td>
<td>6013199</td>
<td>&nbsp;</td>
<tr>
<td>Phosphoric acid solution ( 49-51%, for HPLC)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>79607</td>
<td>&nbsp;</td>
<tr>
<td>Protease Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2714</td>
<td>&nbsp;</td>
<tr>
<td>Protein A-Sepharose beads</td>
<td>&nbsp;</td>
<td>Repligen</td>
<td>IPA300</td>
<td>&nbsp;</td>
<tr>
<td>Rabbit polyclonal anti-H2 carboxy-terminal 6</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sodium deoxycholate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>30970</td>
<td>&nbsp;</td>
<tr>
<td>Sodium dodecyl sulfate</td>
<td>&nbsp;</td>
<td>Bio-RAD</td>
<td>161-0301</td>
<td>&nbsp;</td>
<tr>
<td>Sodium phosphate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>342483</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate solution (100mM)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>Spherisorb NH2 Column, 5 &mu;m, 4.6 x 250 mm</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>PSS831115</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>BDH</td>
<td>306324N</td>
<td>&nbsp;</td>
<tr>
<td>Vibra-Cell ultrasonic processors VCX 750</td>
<td>&nbsp;</td>
<td>Sonics and Materials, Inc.</td>
<td>690-003</td>
<td>&nbsp;</td>
<tr>
<td>Buffer A: 1% Triton X-100, 0.5% w/v sodium deoxycholate, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer B: 0.5% w/v SDS, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer C: N-glycosidase F reaction buffer: 200mM Na3PO4, pH 8.0, 4mM EDTA, 1.5% N-octylglucoside</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NIH 3T3 stably expressing H2a 6</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer D: 0.5% Triton X-100, 0.25% w/v sodium deoxycholate, 0.5% w/v SDS, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer E: 0.5% w/v SDS, 1% v/v 2-Mercaptoethanol, Protease inhibitor cocktail 2% v/v in PBS </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M3148</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

pulse-chase analysis of n-linked sugar chains from glycoproteins in mammalian cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

Watch this Scientific Journal Video about Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells at JoVE.com

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

pulse-chase-analysis-n-linked-sugar-chains-from-glycoproteins

Research

JoVE Journal

Biology

15.1K ビュー.  Tel Aviv University. 我々は、哺乳類細胞におけるそれらの生合成後の糖タンパク質の初期の生活を通じて、N -結合型グリカンの変化の分析のための方法を説明します。これは、代謝的に標識した糖鎖、糖タンパク質およびHPLCによる検討からの酵素遊離のパルス - チェイス分析によって達成されます。

ビデオ: Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by...

analysis of protein folding, transport, and degradation in living cells by radioactive pulse chase

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live...

n-glycan profiling of glycoproteins by hydrophilic interaction liquid chromatography with fluorescence and mass spectrometric detection

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

cycloheximide chase analysis of protein degradation in saccharomyces cerevisiae

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

analysis of cell cycle position in mammalian cells

Analysis of Cell Cycle Position in Mammalian Cells

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol.  We report a simple experimental approach to staining cells and quantifying their position in the cell...

analysis of scap n-glycosylation and trafficking in human cells

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

efficient mammalian cell expression and single-step purification of extracellular glycoproteins for crystallization

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical...

analysis of n-glycans from raphanus sativus cultivars using pngase h+

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

We describe a simple and rapid method for the preparation and analysis of N-glycans from different cultivars of radish (Raphanus...

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

purification of ubiquitinated p53 proteins from mammalian cells

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing...

analysis of mrna nuclear export kinetics in mammalian cells by microinjection

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic...

analysis of dna double-strand break (dsb) repair in mammalian cells

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

mutagenesis and functional analysis of ion channels heterologously expressed in mammalian cells

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the effect of a single point mutation on the function of an ion...

protein complex affinity capture from cryomilled mammalian cells

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic...

production and purification of recombinant antibodies from mammalian cells

The video explains the process of producing antibodies in mammalian host cells, from gene expression to secretion. It involves transcription, translation, signal recognition, correct folding, and eventual release into the extracellular medium via secretory...

Production and Purification of Recombinant Antibodies from Mammalian Cells

Leave the mixture at room temperature for an additional 5 minutes. Add the entire mixture to the CHO cells while manually shaking the flask. Immediately incubate the cells at 37 degrees Celsius while shaking at 130...

immunofluorescence analysis of stress granule formation after bacterial challenge of mammalian cells

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial...

a spin-tip enrichment strategy for simultaneous analysis of n-glycopeptides and phosphopeptides from human pancreatic tissues

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

Post-translational modifications (PTMs) change protein structures and functions. Methods for the simultaneous enrichment of multiple PTM types can maximize coverage in analyses. We present a protocol using dual-functional Ti(IV)-immobilized metal affinity chromatography followed by mass spectrometry for the simultaneous enrichment and analysis of protein N-glycosylation and phosphorylation in pancreatic...

antibody production from a batch overgrowth culture of transfected mammalian cells

The video demonstrates a method for producing therapeutic antibodies using stably transfected mammalian cells. Tryptone supplementation stimulates cell division, resulting in overgrowth and supporting a higher antibody yield. The antibody-containing medium is harvested, filtered, and stored for further...

Antibody Production from a Batch Overgrowth Culture of Transfected Mammalian Cells

tracking sugar-elicited local searching behavior in drosophila

Author Spotlight: Exploring Behavioral Pathways Through Cross-Species Insights in Foraging and Communication

This protocol describes a behavioral assay for recording sugar-elicited search behavior using Drosophila melanogaster. The assay can be utilized to study feeding and foraging-related behaviors, as well as the underlying neuronal...

Tracking Sugar-Elicited Local Searching Behavior in Drosophila

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

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.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...