Denaturing Polyacrylamide Gel Electrophoresis Research Articles

Specific Photocleavage Activity of N‐Methylmesoporphyrin IX on G‐Quadruplex DNA Structures

G‐rich regions, at which G‐quadruplexes (G4) are proposed to form, are associated with promoter regions within proto‐oncogenes and may play a role in cell proliferation and carcinogenesis. The ability to identify the formation of G4 structures in these regions, including the temporal aspects of G‐quadruplex formation, is essential to establish the role of these structures in transcriptional regulation. We have focused on identifying highly selective G4 photocleavage agents as molecular probes for establishing the location and timing of G4 formation. N‐methylmesoporphyrin IX (NMM) has long been known as a G4 ligand; however, there has been little investigation into its DNA photocleavage activity. Here we report the ability of NMM to act as a highly selective photocleavage agent for specific G4 structures. We first studied the interaction of NMM with the G4 structures formed by DNA oligonucleotides containing the c‐MYC promoter or human telomeric (hTel) sequences formed in the presence of either Na+ or K+‐containing buffers using a FRET‐based Tm assay and CD spectroscopy. We employed a fluorescence‐based solution assay for G4 cleavage to establish that NMM effects photocleavage (420 nm) of c‐MYC more efficiently than the hTel G4 under both Na+ and K+ conditions, and effects photocleavage of c‐MYC G4 even under white light irradiation. Using denaturing polyacrylamide gel electrophoresis (PAGE), we confirmed this selective photocleavage and demonstrate that cleavage occurs at specific guanine residues of the c‐MYC G4. In order to determine if the photocleavage proceeds through the generation of reactive oxygen species like singlet oxygen, we carried out photocleavage reactions in the presence of H2O and D2O, and employed a turn‐on fluorescent singlet oxygen sensor to detect 1O2 generation. We find that NMM acts to destabilize specific G4 topologies, but oligonucleotides annealed in the presence of NMM form alternative topologies that are thermally stabilized. NMM photocleavage is highly selective for G4 DNA versus duplex DNA, and the K+‐stabilized c‐MYC G4 is preferentially cleaved compared to the Na+‐stabilized structure and the hTel G4 in either Na+ or K+. PAGE analysis indicates that NMM photocleavage of c‐MYC G4 occurs preferentially at the 3′ G‐tetrad residues. This photocleavage does not appear to be due to 1O2. These results indicate that NMM may be a useful tool for elucidating the role of promoter G‐quadruplex structures.Support or Funding InformationSupport for this work by the Cancer Prevention and Research Institute of Texas (RP160852) and Texas State University is gratefully acknowledged.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A thermostable chitinase from the antagonistic Chromobacterium violaceum that inhibits the development of phytopathogenic fungi

The biocontrol activity of some soil strains of Chromobacterium sp. against pathogenic fungi has been attributed to secreted chitinases. The aim of this work was to characterize biochemically a recombinant chitinase (CvChi47) from C. violaceum ATCC 12472 and to investigate its effects on phytopathogenic fungi. CvChi47 is a modular enzyme with 450 amino acid residues, containing a type I signal peptide at the N-terminal region, followed by one catalytic domain belonging to family 18 of the glycoside hydrolases, and two type-3 chitin-binding domains at the C-terminal end. The recombinant enzyme was expressed in Escherichia coli as a His-tagged protein and purified to homogeneity. The native signal peptide of CvChi47 was used to direct its secretion into the culture medium, from where the recombinant product was purified by affinity chromatography on chitin and immobilized metal. The purified protein showed an apparent molecular mass of 46 kDa, as estimated by denaturing polyacrylamide gel electrophoresis, indicating the removal of the signal peptide. CvChi47 was a thermostable protein, retaining approximately 53.7% of its activity when heated at 100 °C for 1 h. The optimum hydrolytic activity was observed at 60 °C and pH 5. The recombinant chitinase inhibited the conidia germination of the phytopathogenic fungi Fusarium oxysporum and F. guttiforme, hence preventing mycelial growth. Furthermore, atomic force microscopy experiments revealed a pronounced morphological alteration of the cell surface of conidia incubated with CvChi47 in comparison to untreated cells. Taken together, these results show the potential of CvChi47 as a molecular tool to control plant diseases caused by these Fusarium species.

A Simple and Efficient Microfluidic System for Reverse Chemical Synthesis (5′-3′) of a Short-Chain Oligonucleotide Without Inert Atmosphere

Reverse DNA synthesis (5′-3′) plays diverse functional roles in cellular biology, biotechnology, and nanotechnology. However, current microfluidic systems for synthesizing single-stranded DNAs at a laboratory scale are limited. In this work, we develop a simple and efficient polydimethylsiloxane- (PDMS-) based microfluidic system for the reverse chemical synthesis of short-chain oligonucleotides (in the 5′-3′ direction) under ambient conditions. The use of a microfluidics device and anhydrous conditions effectively surpass the problem of moisture sensitivity during oligonucleotide synthesis. With optimized microfluidic synthesis conditions, the system is able to synthesize up to 21 bases-long oligonucleotides in air atmosphere. The as-synthesized oligonucleotides, without further purification, are characterized using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF/TOF) mass spectroscopy (MS) supported by the denatured polyacrylamide gel electrophoresis (PAGE) analysis. This developed system is highly promising for producing the desired sequence at the nanomolar scale on-chip and on-demand in the near future.

Enzymatic Synthesis of Backbone-Modified Oligonucleotides Using T4 DNA Ligase.

T4 DNA ligase in high concentrations of certain crowding agents and cosolutes catalyzes the synthesis of a series of backbone-modified oligonucleotides that are difficult to obtain chemically. Backbone-modified nucleic acids are often enzymatically and chemically more stable, making them interesting as potential diagnostic or therapeutic agents, as a biosafety tool, or in nanotechnology. In this article, we describe a small-scale experiment to probe the efficiency of the ligation reaction of modified oligonucleotides in the presence of 3 M betaine and 10% PEG 8000, followed by large-scale ligation with subsequent isolation of the ligated oligonucleotide. The correct product formation can be verified using denaturing polyacrylamide gel electrophoresis and mass spectrometry. © 2019 by John Wiley & Sons, Inc.

HETEROGENEITY OF PROTEINS IN BIRDS’ EGG-WHITES

Little is known about the comparative heterogeneity of protein compounds among a wide variety of birds’ egg white, particularly, an analysis of their detailed, in-parallel protein composition. Hence this research is conducted mainly to evaluate the extent of variability among 42 types of birds’ egg white. To improve the perception of these biological fluids, the main phenotypic variations of egg whites were evaluated using the discontinuous denaturing polyacrylamide gel electrophoresis (SDS-PAGE), Gradient SDS-PAGE, Native-PAGE, cellulose acetate electrophoresis, and the reverse-phase high-performance liquid chromatography (RP-HPLC). The results showed that the Native-PAGE and SDS-PAGE produced better screening results than other methods in identifying protein. Although  Native-PAGE and SDS-PAGE did not show remarkable variability in terms of hydrophobicity, several electrophoretic differences of egg-white proteins were observed. Several unknown proteins in the egg white samples of different bird species were also identified through the electrophoretic experiments. Hence, it might be possible, as in the case of egg white samples, to provide a characterized assessment among birds by using only the available gel electrophoresis techniques. This study also provided a rapid snapshot of the initial identification of several unknown components of egg white proteins. Accordingly, this study constituted the first large-scale comparative proteomics investigation performed among the largely varying types of egg whites from commercial stores and bird keepers in the middle Euphrates areas in Iraq.

In vitro RNA Cleavage Assays to Characterize IRE1-dependent RNA Decay.

The kinase/RNase IRE1 is a key effector of the cellular response to endoplasmic reticulum stress. The RNase activity of IRE1 can be measured in cells or in the test tube. Here we describe a protocol for the in vitro cleavage and analysis of RNA substrates of IRE1. The method consists of the in vitro transcription, purification and re-folding of IRE1 substrate RNAs followed by their cleavage using recombinant cytosolic kinase/RNase domains of IRE1 and the separation of the resulting fragments by denaturing polyacrylamide gel electrophoresis. This protocol allows the study of the cleavage kinetics of IRE1’s RNA substrates in vitro.

3'-Linker Ligation and Size Selection by SDS-PAGE for Cross-Linking Immunoprecipitation (CLIP).

This protocol describes the purification by denaturing polyacrylamide gel electrophoresis of RNA linkers for cross-linking immunoprecipitation (CLIP). Purification is necessary because if the 3' linker loses the puromycin blocking group, concatemerization of the 3' linker will occur during the 3' linker ligation reaction. In addition, truncated linkers make bioinformatic processing of the sequencing results more difficult than it need be. Additionally, this protocol describes the treatment of coimmunoprecipitated RNA tags for CLIP with alkaline phosphatase to remove the 3' phosphate remaining after RNase digestion. Dephosphorylation prevents intramolecular circularization of RNA during subsequent ligation to the linker. The purified RNA linker, blocked with puromycin at its 3' end to prevent linker-linker multimerization, is then ligated to the 3' end of the RNA tag. Removal of free linker is accomplished by performing the ligation while the RNABP:RNA complex is associated, via antibody, to protein A Dynabeads, allowing thorough washing and linker removal. Additional purification is achieved by SDS-PAGE and transfer of the size-selected RNABP:RNA complexes to nitrocellulose.

Identification of Glycoproteins Isolated from Extracellular Polymeric Substances of Full-Scale Anammox Granular Sludge.

ANaerobic AMMonium OXidation (anammox) is an established process for efficient nitrogen removal from wastewater, relying on anammox bacteria to form stable biofilms or granules. To understand the formation, structure, and stability of anammox granules, it is important to determine the composition of the extracellular polymeric substances (EPS). The aim of this research was to elucidate the nature of the proteins, which are the major fraction of the EPS and were suspected to be glycosylated. EPS were extracted from full-scale anammox granular sludge, dominated by “Candidatus Brocadia”, and subjected to denaturing polyacrylamide gel electrophoresis. By further analysis with mass spectrometry, a high abundant glycoprotein, carrying a heterogeneous O-glycan structure, was identified. The potential glycosylation sequence motif was identical to that proposed for the surface layer protein of “Candidatus Kuenenia stuttgartiensis”. The heavily glycosylated protein forms a large fraction of the EPS and was also located by lectin staining. Therefore, we hypothesize an important role of glycoproteins in the structuring of anammox granules, comparable to the importance of glycans in the extracellular matrix of multicellular organisms. Furthermore, different glycoconjugates may have distinct roles in the matrix of granular sludge, which requires more in-depth characterization of different glycoconjugates in future EPS studies.

The Application of Molecular Markers to Accelerate the Recovery of Cytoplasmic and Nuclear Male Sterility in South African Onion (Allium cepa L.) Hybrid Parental Lines

Male sterility is important to prevent self-pollination and loss of the onion hybrid genotype. Classic methods require 4-8 years of progeny testing before the cytoplasm type can be determined. An accurate and time-saving method was needed. Various types of markers were tested for application to South African onion parental lines of hybrid cultivars, and which could determine male sterility and maintainer genotypes accurately and easily with large numbers of samples. Five cytoplasmic (5’cob, orfA501, orf725, IGS and cob-type 2) and four nuclear markers (jnurf13, isotig34671_610, isotig30856_1351 and isotig29186_1830) were sourced. Genomic DNA was isolated from onion seedlings and young leaves growing from bulbs in the Agricultural Research Council (ARC) research field. PCR marker amplification products were separated by agarose or denaturing polyacrylamide gel electrophoresis (PAGE) gels. Real-time polymerase chain reaction (PCR) was performed with custom TaqMan® SNP genotyping assays containing primer/probe pairs designed to detect single nucleotide polymorphisms (SNPs) linked to the nuclear Ms locus. OrfA501 proved useful as a presence/absence marker for cytoplasmic male sterility, while TaqMan® SNP genotyping assays were superior to the jnurf13 nuclear marker in terms of rapid throughput. PCR molecular markers and custom TaqMan® SNP genotyping assays were efficient in screening the onion lines rapidly and accurately for their cytoplasmic and nuclear male sterility genotype. These methods reduced the time to identify the correct genotype of male sterile and maintainer lines, gave accurate genotypic information and proved to be useful on a larger scale. These molecular marker methods will facilitate the production of the correct seed for commercialization of onion lines worldwide.

Bifunctional cross-linking approaches for mass spectrometry-based investigation of nucleic acids and protein-nucleic acid assemblies.

With the goal of expanding the very limited toolkit of cross-linking agents available for nucleic acids and their protein complexes, we evaluated the merits of a wide range of bifunctional agents that may be capable of reacting with the functional groups characteristic of these types of biopolymers. The survey specifically focused on the ability of test reagents to produce desirable inter-molecular conjugates, which could reveal the identity of interacting components and the position of mutual contacts, while also considering a series of practical criteria for their utilization as viable nucleic acid probes. The survey employed models consisting of DNA, RNA, and corresponding protein complexes to mimic as close as possible typical applications. Denaturing polyacrylamide gel electrophoresis (PAGE) and mass spectrometric (MS) analyses were implemented in concert to monitor the formation of the desired conjugates. In particular, the former was used as a rapid and inexpensive tool for the efficient evaluation of cross-linker activity under a broad range of experimental conditions. The latter was applied after preliminary rounds of reaction optimization to enable full-fledged product characterization and, more significantly, differentiation between mono-functional and intra- versus inter-molecular conjugates. This information provided the feedback necessary to further optimize reaction conditions and explain possible outcomes. Among the reagents tested in the study, platinum complexes and nitrogen mustards manifested the most favorable characteristics for practical cross-linking applications, whereas other compounds provided inferior yields, or produced rather unstable conjugates that did not survive the selected analytical conditions. The observed outcomes will help guide the selection of the most appropriate cross-linking reagent for a specific task, whereas the experimental conditions described here will provide an excellent starting point for approaching these types of applications. As a whole, the results of the survey clearly emphasize that finding a universal reagent, which may afford excellent performance with all types of nucleic acid substrates, will require extending the exploration beyond the traditional chemistries employed to modify the constitutive functional groups of these vital biopolymers.

Serendipitous Discovery of a Guanine-rich DNA Molecule with a Highly Stable Structure in Urea

We have made an accidental discovery of an unusual, single-stranded, guanine-rich DNA molecule that is capable of adopting a folded structure in 7 M urea (7MU) known to denature nucleic acid structures. The folding of this molecule requires Na+ and Mg2+ and the folded structure remains stable when subjected to denaturing (7MU) polyacrylamide gel electrophoresis. Results from sequence mutagenesis, DNA methylation, and circular dichroism spectroscopy studies suggest that this molecule adopts an intramolecular guanine-quadruplex structure with 5 layers of guanine tetrads. Our finding indicates that DNA has the ability to create extremely stable structural folds despite its limited chemical repertoire, making it possible to develop DNA-based systems for unconventional applications.

Contribution of linoleic acid to the formation of advanced glycation end products in model systems during heat treatment

Advanced glycation end products (AGEs) are glycosylated metabolic products generated in vivo and are associated with aging-related diseases. They are also formed during heat treatment in food processing. In this work, we investigated the contribution of linoleic acid (LA) to AGE formation using a protein/glucose model. An electronic tongue, denaturing polyacrylamide gel electrophoresis, electron spin resonance spectroscopy, circular dichroism, and ultraperformance liquid chromatography-tandem mass spectrometry were used to analyse reaction intermediates and reactive radical formation. The results show that LA is the key factor responsible for the change in flavour including the rapid triggering of glycation reactions. The amount of lipid-induced reactive radicals was significantly higher than in the non-fat system, radical generation in the non-fat system was gradually quenched after a robust radical-yielding reaction in the first 25 minutes. Subsequent unsaturated lipid oxidation, and AGE accumulation surpass Maillard reaction-only outcomes. Initial LA-induced changes in protein structure are followed by glycation and are enhanced by hydrophobic interactions and increased carbonyl levels resulting from lipid oxidation. These findings implicate lipids and lipid oxidation as the main factors responsible for AGE formation during the processing of fat-rich unsaturated fatty acid-containing foods.

The Nonbulky DNA Lesions Spiroiminodihydantoin and 5-Guanidinohydantoin Significantly Block Human RNA Polymerase II Elongation in Vitro.

The most common, oxidatively generated lesion in cellular DNA is 8-oxo-7,8-dihydroguanine, which can be oxidized further to yield highly mutagenic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in DNA. In human cell-free extracts, both lesions can be excised by base excision repair and global genomic nucleotide excision repair. However, it is not known if these lesions can be removed by transcription-coupled DNA repair (TCR), a pathway that clears lesions from DNA that impede RNA synthesis. To determine if Sp or Gh impedes transcription, which could make each a viable substrate for TCR, either an Sp or a Gh lesion was positioned on the transcribed strand of DNA under the control of a promoter that supports transcription by human RNA polymerase II. These constructs were incubated in HeLa nuclear extracts that contained active RNA polymerase II, and the resulting transcripts were resolved by denaturing polyacrylamide gel electrophoresis. The structurally rigid Sp strongly blocks transcription elongation, permitting 1.6 ± 0.5% nominal lesion bypass. In contrast, the conformationally flexible Gh poses less of a block to human RNAPII, allowing 9 ± 2% bypass. Furthermore, fractional lesion bypass for Sp and Gh is minimally affected by glycosylase activity found in the HeLa nuclear extract. These data specifically suggest that both Sp and Gh may well be susceptible to TCR because each poses a significant block to human RNA polymerase II progression. A more general principle is also proposed: Conformational flexibility may be an important structural feature of DNA lesions that enhances their transcriptional bypass.

Electrophoretic Separation of Humic Acids Isolated from Tropical Soils Through Modified Denaturing Polyacrylamide Gel Electrophoresis

Electrophoretic separation of humic acids obtained from soil was attempted through denaturing polyacrylamide gel electrophoresis (dn-PAGE) by modifying the known methods. The modifications of sample preparation and gel composition led to significant improvement in both visibility and intensity of humic acid profile. The comparative profile showed a similar nature of humic acid components in all the samples and the commercial standard. The Fourier transform infrared spectroscopy (FTIR) spectra indicated the presence of carboxyl and carbonyl functional groups and aliphatic chains in humic acid samples. The modified method facilitated better separation of humic acid. The results of the present study indicate the potential of dn-PAGE for electrophoretic separation of different components of humic acids.

Virtual Cross-Linking of the Active Nemorubicin Metabolite PNU-159682 to Double-Stranded DNA.

The DNA alkylating mechanism of PNU-159682 (PNU), a highly potent metabolite of the anthracycline nemorubicin, was investigated by gel-electrophoretic, HPLC-UV, and micro-HPLC/mass spectrometry (MS) measurements. PNU quickly reacted with double-stranded oligonucleotides, but not with single-stranded sequences, to form covalent adducts which were detectable by denaturing polyacrylamide gel electrophoresis (DPAGE). Ion-pair reverse-phase HPLC-UV analysis on CG rich duplex sequences having a 5'-CCCGGG-3' central core showed the formation of two types of adducts with PNU, which were stable and could be characterized by micro-HPLC/MS. The first type contained one alkylated species (and possibly one reversibly bound species), and the second contained two alkylated species per duplex DNA. The covalent adducts were found to produce effective bridging of DNA complementary strands through the formation of virtual cross-links reminiscent of those produced by classical anthracyclines in the presence of formaldehyde. Furthermore, the absence of reactivity of PNU with CG-rich sequence containing a TA core (CGTACG), and the minor reactivity between PNU and CGC sequences (TACGCG·CGCGTA) pointed out the importance of guanine sequence context in modulating DNA alkylation.

Assembling RNA Nanoparticles.

RNA nanoparticles are designed and self-assembled according to noncanonical interactions of naturally conserved RNA motifs and/or canonical Watson-Crick base-pairing interactions, which have potential applications in gene therapy and nanomedicine. These artificially engineered nanoparticles are mainly synthesized from in vitro transcribed RNAs, purified by denaturing and native polyacrylamide gel electrophoresis (PAGE), and characterized with native PAGE, AFM, and TEM technologies. The protocols of in vitro transcription, denaturing and native PAGE, and RNA nanoparticle self-assembly are described in detail.

Cirrhosis related functionality characteristic of the fecal microbiota as revealed by a metaproteomic approach.

BackgroundIntestinal microbiota operated as a whole and was closely related with human health. Previous studies had suggested close relationship between liver cirrhosis (LC) and gut microbiota.MethodsTo determine the functional characteristic of the intestinal microbiota specific for liver cirrhosis, the fecal metaproteome of three LC patients with Child-Turcotte-Pugh (CTP) score of A, B, and C, and their spouse were first compared using high-throughput approach based on denaturing polyacrylamide gel electrophoresis and liquid chromatography–tandem mass spectrometry in our study.ResultsA total of 5,020 proteins (88 % from bacteria, 12 % form human) were identified and annotated based on the GO and KEGG classification. Our results indicated that the LC patients possessed a core metaproteome including 119 proteins, among which 14 proteins were enhanced expressed and 7 proteins were unique for LC patients compared with the normal, which were dominant at the function of carbohydrate metabolism. In addition, LC patients have unique biosynthesis of branched chain amino acid (BCAA), pantothenate, and CoA, enhanced as CTP scores increased. Those three substances were all important in a wide array of key and essential biological roles of life.ConclusionsWe observed a highly comparable cirrhosis-specific metaproteome clustering of fecal microbiota and provided the first supportive evidence for the presence of a LC-related substantial functional core mainly involved in carbohydrate, BCAA, pantothenate, and CoA metabolism, suggesting the compensation of intestinal microbiota for the fragile and innutritious body of cirrhotic patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12876-016-0534-0) contains supplementary material, which is available to authorized users.

The influence of diisocyanate antigen preparation methodology on monoclonal and serum antibody recognition

ABSTRACTExposure to diisocyanates (dNCOs), such as methylene diphenyl diisocyanate (MDI) can cause occupational asthma (OA). Currently, lab tests for dNCO specific IgE are specific, but not sensitive, which limits their utility in diagnosing dNCO asthma. This may be due to variable preparation and poor characterization of the standard antigens utilized in these assays. The aim of this study was to produce and characterize a panel of antigens prepared using three different commonly employed methods and one novel method. The conjugates were examined for recognition by anti-MDI monoclonal antibodies (mAbs) in varying enzyme linked immunosorbant assay (ELISA) formats, extent of crosslinking, total amount of MDI, the sites of MDI conjugation, relative shape/charge, and reactivity with human serum with antibodies from sensitized, exposed workers. Results indicate that while there are minimal differences in the total amount of MDI conjugated, the extent of crosslinking, and the conjugation sites, there are significant differences in the recognition of differently prepared conjugates by mAbs. Native and denaturing polyacrylamide gel electrophoresis demonstrate differences in the mobility of different conjugates, indicative of structural changes that are likely important for antigenicity. While mAbs exhibited differential binding to different conjugates, polyclonal serum antibodies from MDI exposed workers exhibited equivalent binding to different conjugates by ELISA. While differences in the recognition of the different conjugates exist by mAb detection, differences in antigenicity could not be detected using human serum from MDI-sensitized individuals. Thus, although dNCO conjugate preparation can, depending on the immunoassay platform, influence binding of specific antibody clones, serologic detection of the dNCO-exposure-induced polyclonal antibody response may be less sensitive to these differences.

Antimicrobial Protein Candidates from the Thermophilic Geobacillus sp. Strain ZGt-1: Production, Proteomics, and Bioinformatics Analysis.

A thermophilic bacterial strain, Geobacillus sp. ZGt-1, isolated from Zara hot spring in Jordan, was capable of inhibiting the growth of the thermophilic G. stearothermophilus and the mesophilic Bacillus subtilis and Salmonella typhimurium on a solid cultivation medium. Antibacterial activity was not observed when ZGt-1 was cultivated in a liquid medium; however, immobilization of the cells in agar beads that were subjected to sequential batch cultivation in the liquid medium at 60 °C showed increasing antibacterial activity up to 14 cycles. The antibacterial activity was lost on protease treatment of the culture supernatant. Concentration of the protein fraction by ammonium sulphate precipitation followed by denaturing polyacrylamide gel electrophoresis separation and analysis of the gel for antibacterial activity against G. stearothermophilus showed a distinct inhibition zone in 15–20 kDa range, suggesting that the active molecule(s) are resistant to denaturation by SDS. Mass spectrometric analysis of the protein bands around the active region resulted in identification of 22 proteins with molecular weight in the range of interest, three of which were new and are here proposed as potential antimicrobial protein candidates by in silico analysis of their amino acid sequences. Mass spectrometric analysis also indicated the presence of partial sequences of antimicrobial enzymes, amidase and dd-carboxypeptidase.

Different cells make different proteins: a laboratory exercise illustrating tissue-specific protein expression in animals

All the cells of higher organisms have the same DNA but not the same proteins. Each type of specialised cell that forms a tissue has its own pattern of gene expression and, consequently, it contains a particular set of proteins that determine its function. Here, we describe a laboratory exercise addressed to undergraduate students that aims to reinforce the following concept: what makes cells different is they make different proteins. The students performed a denaturing polyacrylamide gel electrophoresis (SDS–PAGE) to separate the proteins extracted from different animal tissues, and then obtained and compared the corresponding protein profiles. In addition, the students performed a Western blot analysis to detect the presence or absence of a tissue-specific protein in the different tissue extracts. This laboratory exercise allowed students to understand the basis of the cell differentiation better and also provided them the opportunity to learn a variety of analytical laboratory techniques.