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Protein Array Protocols Protein Array protokoller	Protein Array Bioinformatics Protein Array Bioinformatik	Learn about Protein Arrays Lær om Protein Arrays	Protein Array Kits and Products Protein Array Kits og Produkter	Protein Array Forum Protein Array Forum	Proteomic News Proteomiske Nyheder

Protein Microarray Chips Protein microarray Chips

Protein and Antibody Microarrays Protein og Antibody Microarrays

Table of Contents Indholdsfortegnelse

Introduction and Background to Protein and Antibody Microarrays Indledning og baggrund for protein og Antibody Microarrays

Types of Antibody and Protein Chips Typer af antistof og Protein Chips

Protein and Antibody Attachment Methods - Creation of a Microarray Chip Protein og Antibody Attachment Metoder - Oprettelse af et microarray Chip

Protein Chip Delivery Methods Protein Chip Delivery Methods

Protein Chip Capture Molecules and Their Limitations Protein Chip Capture Molekyler og deres begrænsninger

Antibody Microarrays: Problems and Solutions Antibody Microarrays: problemer og løsninger

Protein Microarray Detection Methods and Analysis Protein microarray påvisningsmetoder og Analyse

Protein Production for Protein Arrays Protein Production for Protein Arrays

Applications of Protein Arrays and Protein Chips Anvendelser af Protein Arrays og Protein Chips

Protein Microarrays: Future Directions and Conclusions Protein Microarrays: Fremtidige retninger og konklusioner

References for Protein and Antibody Microarrays Referencer for protein og Antibody Microarrays

Introduction and Background to Protein and Antibody Microarrays. Indledning og baggrund for protein og Antibody 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). På trods af nylige fremskridt i vores forståelse af molekylær biologi, i mange tilfælde er vi ude af stand til at involvere bestemte proteiner med en sygdom. Genomforskning og microarray teknologi har gjort det muligt for os at analysere tusindvis af mRNAs på én gang, og afgøre, om mRNA ekspression er ændret i sygdom stater. Men forskere har længe vidst, at koncentrationen af en mRNA inden for en celle er dårligt korreleret med den faktiske forekomst af dette 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). Dette skyldes, at satsen for nedbrydning af individuelle mRNAs og proteiner adskiller sig, post-transskriptionel kontrol af protein oversættelse (4), et antal post-transskriptionel ændringer af protein (5), og protein nedbrydning ved proteolyse (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. Ved at måle størrelsen af den specifikke protein direkte, vi måler et rigtigt niveau af genet funktion. Men når man tager i betragtning af det store antal post-translationel ændringer, menneskelige celler kan indeholde en million eller flere forskellige protein varianter, nogen af som kan ændres inden sygdommen gør opgaven med at analysere dem alle en enorm opgave.  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. Protein microarrays eller protein chips mai give mulighed for en løsning på dette problem. Et dias eller "chip" kunne være plettet med tusindvis af kendte antistoffer eller peptider som en DNA microarray, en biologisk prøve spredt over chippen, og alle bindende bestemmes.  Binding could also be analyzed using standard proteomic techniques such as time-of-flight mass spectrometry (MS) and peptide mass fingerprinting. Bindende også kunne analyseres ved hjælp af standard proteomiske teknikker såsom time-of-flight mass spectrometry (MS) og peptid masse fingeraftryk.  Protein chips can thus become a fast and high-throughput method of profiling protein changes in disease. Protein-chips kan således blive en hurtig og høj overførselshastighed metoden med profilering protein ændringer i sygdommen. (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 har potentiale til at fungere i mange andre anvendelser, herunder en undersøgelse af protein-protein, protein-lægemiddelinteraktioner, DNA-protein interaktioner, protein localization, antigen-antistof interaktioner, enzym-substrat, og receptor-ligand interaktioner som alle mai kunne ændres til array-typen high-throughput screening (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). To tilgange er blevet anvendt til at karakterisere flere proteiner i en biologisk prøve. Den første strategi er 2-dimensional gelelektroforese, som har været meget anvendt til at sortere og visualisere op til 2000-10000 proteiner i et enkelt eksperiment ved excision og identifikation af massespektrometri (MS) (9). Denne metode er både tidskrævende og endda med MS, er det kun de mest rigelige proteiner kan afsløres. Also, reproducerbarhed er problematisk, selv om pre-cast geler og almindeligt anvendte reagenser, protokoller og hardware komponenter har ført til forbedret ydeevne (17). På grund af begrænsningerne af 3D-gel adskillelse teknologi, øget opmærksomhed på, er at fokusere på udviklingen af den anden tilgang, udvikling af protein microarrays som et alternativ og supplerende tilgang (10-12).
The theoretical background for protein microarray-based ligand binding assays was initially developed by Ekins et al. Den teoretiske baggrund for protein microarray-baserede ligand bindende assays blev oprindeligt udviklet af 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). i slutningen af 1980'erne (13-16). Ifølge den model, antistof microarrays ikke kun vil tillade samtidige screening af en analysand panel, men ville også være mere følsom og hurtigere end konventionelle screeningsmetoder. Interessen for screening store protein indeholder kun opstod som et resultat af resultater inden for genomforskning af DNA microarrays og det menneskelige genom Project (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). Den første array tilgange forsøgt at miniaturize biokemiske og immunbiologiske assays normalt udføres i 96-brønds mikrotiterplader (18-19). 96-brønds antistof arrays blev først oprettet med 144 elementer hver for standard enzyme-linked immunosorbent assays (ELISA) (20) . Lignende arrays blev brugt til at måle prostata-specifikke antigen (PSA) og cytokiner (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 membraner også blev oprindeligt brugt på grund af deres overlegne proteinbinding kapacitet. De var for det meste probed med antistoffer ved hjælp af ELISA-teknik. En lav massefylde array af 48 rensede proteiner involveret i transskription blev udviklet til undersøgelse af specifikke interaktioner af proteiner med radioaktivt DNA, RNA , Ligands, og andre små kemikalier (22). A membran-baserede high density array blev udviklet med henblik på screening en menneskelig fetal brain cDNA udtryk bibliotek bestående af 37830 kloner. Renset proteiner blev spottet på PVDF membraner med en tæthed på 300 prøver / cm2 (23). Andre filter baseret arrays er konstrueret, men de begrænsninger var lav opløsning og betydelige baggrund, som gør det vanskeligt at bruge dem i programmer med begrænsning prøve mængder såsom protein udtryk profilering af svulst biopsier.

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 arrays er kompromitteret af et bibliotek af proteiner eller antistoffer immobiliseret i en 2D adresserbare grid på en chip (se figur 1). Protein microarray biochips ekstrakt og fastholde målene fra flydende medier og adskiller sig fra microfluidic biochips, som adskiller og bearbejde proteiner i en transport medium in situ ved hjælp microfluidic anordninger (24,25). En typisk array kan indeholde 103-104 rumligt adskilte elementer inden for et samlet areal på 1 cm2 (26).

Next: Types of Antibody and Protein Chips Næste: Typer af antistof og Protein Chips

References for Protein and Antibody Microarrays Referencer for protein og Antibody Microarrays