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Protein Array Protocols Protein Array Protocols	Protein Array Bioinformatics Protein Array bioinformatiku	Learn about Protein Arrays Saznajte više o protein Razvrstati	Protein Array Kits and Products Protein Array Kits i proizvodi	Protein Array Forum Protein Array Forum	Proteomic News Proteomic Novosti

Protein Microarray Chips Protein Microarray Chips

Protein and Antibody Microarrays Protein i Antitijelo Microarrays

Table of Contents Table of Contents

Introduction and Background to Protein and Antibody Microarrays Uvod i pozadina na protein i Antitijelo Microarrays

Types of Antibody and Protein Chips Vrste Antitijelo i protein Chips

Protein and Antibody Attachment Methods - Creation of a Microarray Chip Protein i Antitijelo Priloga Metode - za izradu jednog Microarray Chip

Protein Chip Delivery Methods Protein Chip Način dostave

Protein Chip Capture Molecules and Their Limitations Protein Chip Capture molekula i njihove granice

Antibody Microarrays: Problems and Solutions Antitijelo Microarrays: problemi i rješenja

Protein Microarray Detection Methods and Analysis Protein Microarray Metode i Detection Analysis

Protein Production for Protein Arrays Protein-protein Proizvodnja za Razvrstati

Applications of Protein Arrays and Protein Chips Aplikacije Razvrstati od proteina i protein Chips

Protein Microarrays: Future Directions and Conclusions Protein Microarrays: Budućnost Upute i zaključci

References for Protein and Antibody Microarrays Reference za protein i Antitijelo Microarrays

Introduction and Background to Protein and Antibody Microarrays. Uvod i pozadina na protein i Antitijelo Microarrays.

In spite of recent advancements in our understanding of molecular biology, in many cases we are unable to implicate specific proteins with a disease.  Genomics and microarray technology have allowed us to analyze thousands of mRNAs at one time and determine whether mRNA expression is changed in disease states.  However, researchers have long known that the concentration of an mRNA within a cell is poorly correlated with the actual abundance of that protein (1,2,3). Unatoč nedavnim advancements u naše razumijevanje molekularne biologije, u mnogim slučajevima nismo u mogućnosti obuhvatiti specifični proteini s bolesti. Genomiku i microarray tehnologije su dozvoljeni nas za analizu tisuća mRNAs jedno vrijeme i utvrditi da li je izražaj mRNA promjena u bolesti države. Međutim, znanstvenici su dugo poznati da je koncentracija od mRNA unutar stanica je slabo korelirani sa stvarnim obilja da protein (1,2,3).  This is due to the fact that the rate of degradation of individual mRNAs and proteins differ, post-transcriptional control of protein translation (4), a number of post-transcriptional modifications of protein (5), and protein degradation by proteolysis (6). To je zbog činjenice da je stopa degradacija pojedinih mRNAs i proteini se razlikuju, post-transcriptional control of protein prijevod (4), broj post-transcriptional izmjene protein (5), i degradacija proteina po proteolysis (6) .
By measuring the amount of the specific protein directly, we are measuring a true level of gene function.  However, when one takes into consideration the large number of post-translational modifications, human cells may contain a million or more different protein variants, any of which could be altered in disease making the task of analyzing all of them a huge task. By measuring iznos od specifičnih proteina direktno, mi smo pravi mjerenje razine funkcija gena. Međutim, kada jedan uzima u obzir veliki broj post-prijevodna modifikacije, ljudskih stanica svibanj sadrži milijun ili više različitih varijanti proteina, bilo koji od koja bi se mogla mijenjati u bolest što je zadatak da analizira sve njih veliki zadatak.  Protein microarrays or protein chips may allow for a solution to this problem.  A slide or "chip" could be spotted with thousands of known antibodies or peptides like a DNA microarray, a biological sample spread over the chip, and any binding determined. Microarrays protein ili protein chips svibanj dopustiti za rješenje ovog problema. Slide ili "chip" bi mogao biti uočena s tisućama poznatih protutijela ili peptida poput DNA microarray, biološki uzorak proširila preko čip, i bilo koji određuju obvezujući.  Binding could also be analyzed using standard proteomic techniques such as time-of-flight mass spectrometry (MS) and peptide mass fingerprinting. Obvezujuća također mogao biti analizirani pomoću standardnih proteomic tehnika kao što su vrijeme-od-masena spektrometrija (MS) i peptida masa fingerprinting.  Protein chips can thus become a fast and high-throughput method of profiling protein changes in disease. Protein chips može postati tako brzo i visoko-propusna moć metoda profiliranje proteina promjene u bolesti. (7)

Protein chips have the potential to function in many other applications including the study of protein–protein, protein–drug interactions, DNA-protein interactions, protein localization, antigen-antibody interactions, enzyme-substrate, and receptor-ligand interactions all of which may be amendable to array-type high-throughput screening (7,8). Protein chips imaju potencijal da funkcija u mnogim drugim aplikacijama, uključujući proučavanje protein-protein, protein-droga interakcije, DNA-protein interakcije, lokalizacija proteina, antigen-antitijelo interakcije, enzim-podloga, a receptor-ligand interakcija svih koji svibanj biti amendable na polje tipa visoke propusnosti probira (7,8).

Two approaches have been used in order to characterize multiple proteins in a biological sample.  The first approach is 2-dimensional gel electrophoresis, which has been widely used to separate and visualize up to 2000-10,000 proteins in a single experiment by excision and identification by mass spectrometry (MS) (9).  This method is both time consuming and even with MS, only the most abundant proteins can be detected.  Also, reproducibility is problematic, even though pre-cast gels and commonly used reagents, protocols, and hardware components have led to improved performance (17).  Due to the limitations of 3D-gel separation technology, increasing attention is focusing on the development of the second approach, the development of protein microarrays as an alternative and complementary approach (10-12). Dva pristupa su korišteni da bi karakterizirati više proteina u biološki uzorak. Prvi pristup je 2-dimenzionalan gel-elektroforeza, koji je naširoko koristi za odvajanje i zamišljati do 2000-10000 proteina u jednom eksperimentu by excision i identifikacije koju masena spektrometrija (MS) (9). Ova metoda je i vremena i čak s MS, samo najbitnije bogata proteinima može biti otkriven. Također, reproducibilnost je problematičan, iako pre-cast gelovi i uobičajeno Reagensi, protokoli, hardver i komponente su doveli do poboljšanih performansi (17). Zbog ograničenja 3D-gel tehnologijom odvajanja, čime se povećava pažnja je usmjerena na razvoj i drugi pristup, razvoj protein microarrays kao alternativa i komplementarni pristup (10-12).
The theoretical background for protein microarray-based ligand binding assays was initially developed by Ekins et al. Teorijske pozadine za microarray protein-based ligand binding eseji je u početku developed by Ekins et al. in the late 1980s (13-16).  According to the model, antibody microarrays not only would permit simultaneous screening of an analyte panel, but would also be more sensitive and rapid than conventional screening methods.  Interest in screening large protein sets only arose as a result of the achievements in genomics by DNA microarrays and the Human Genome Project (17). u kasnom 1980s (13-16). Prema model, antitijela microarrays ne samo da bi dozvola za simultano screening of analyte jednoj ploči, ali bi također biti osjetljive i brze nego konvencionalne metode probira. interesa u screening veliki protein postavlja samo ustade kao rezultat postignuća u genomics by DNA microarrays i ljudski genom projekt (17).

The first array approaches attempted to miniaturize biochemical and immunobiological assays usually performed in 96-well microtiter plates (18-19).  96-well antibody arrays were first created with 144 elements each for standard enzyme-linked immunosorbent assays (ELISAs) (20).  Similar arrays were used to measure prostate-specific antigen (PSA) and cytokines (21). Prvi polje pristupi pokušali miniaturize biokemijski i immunobiological eseji obično izvodi u 96-i microtiter plates (18-19). 96-antitijela i nizovi su prvi urednik sa 144 elemenata za svaki standard enzim-linked immunosorbent eseji (ELISAs) (20) . Slične polja su bila korištena za mjerenje prostata-specifični antigen (PSA) i citokini (21).

Filter membranes were also initially used because of their superior protein binding capacity.  They were mostly probed with antibodies using ELISA techniques.  A low density array of 48 purified proteins involved in transcription was developed for the investigation of specific interactions of proteins with radiolabeled DNA, RNA, ligands, and other small chemicals (22).  A membrane-based high density array was developed for the purpose of screening a human fetal brain cDNA expression library consisting of 37830 clones.  Purified proteins were spotted onto PVDF membranes at a density of 300 samples/cm2 (23).  Other filter based arrays were constructed but the limitations were the low resolution and considerable background making it difficult to use them in applications with limiting sample quantities such as protein expression profiling of tumor biopsies. Filter membrane su također u početku koriste zbog njihove superiorne protein binding kapaciteta. Oni su bili uglavnom probed s protutijela pomoću ELISA tehnika. Niske gustoće polje očistili od 48 proteina koji su uključeni u transkripcije je razvijen za istraživanje specifične interakcije proteina s radiolabeled DNA, RNA , Liganada i druge male kemikalije (22). A membrane-based visoke gustoće polje je razvijen u svrhu probira ljudskim fetalni mozak cDNA library izraz se sastoji od 37830 klonovi. Očistili proteini su uočena na PVDF membrane u gustoći od 300 uzoraka / cm2 (23). Other filtar temelji polja su pripremljeni, ali ograničenja su niske rezolucije i znatnu pozadinu, čineći ga teško koristiti ih u aplikacijama s ograničavanja uzorak količine proteina, kao što su izraz profiliranje tumora biopsies.

Protein arrays are compromised of a library of proteins or antibodies immobilized in a 2D addressable grid on a chip (see Figure 1).  Protein microarray biochips extract and retain targets from liquid media and are distinct from microfluidic biochips, which separate and process proteins in a transport medium in situ using microfluidic devices (24,25).  A typical array may contain 103-104 spatially distinct elements within a total area of 1 cm2 (26). Protein polja su ugrožena od knjižnica proteina ili protutijela immobilized u 2D adresabilni grid na čip (vidi Slika 1). Protein microarray biochips izvuci i zadržati ciljeva iz medija i liquid su različita od microfluidic biochips, koji odvojena procesa i proteina u transport medium in situ koristeći microfluidic uređaji (24,25). Tipični polje svibanj sadržavati 103-104 prostorno različite elemente u roku od ukupne površine 1 cm2 (26).

Next: Types of Antibody and Protein Chips Next: Vrste Antitijelo i protein Chips

References for Protein and Antibody Microarrays Reference za protein i Antitijelo Microarrays