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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 Detection Methods and Analysis Protein Microarray Metode i Detection Analysis

Protein and Antibody Microarrays Protein i Antitijelo Microarrays

Detection Methods and Analysis for Protein and Antibody Chips Metode za otkrivanje i analiza proteina i Antitijelo Chips

Detection Methods and Non-specific Binding Metode za otkrivanje i non-posebna obvezujuća
Non-specific binding to the array needs to be minimized and this is typically done by immersing the arrays in a bovine serum albumin based buffer (BSA) (31). Non-posebna obvezujuća u polje treba biti minimiziran i to je obično činio by immersing i polja u goveđi serum albumin temelji međuspremnik (BSA) (31).
Analyte-binding and retention on protein arrays proceeds via thermodynamically driven binding mechanism similar to the hybridization of nucleic acid targets to probes.  However, the detection of bound targets to proteins is considerably more complex than that of DNA microarray detection (9).  Currently a variety of detection methods are being examined.  For example, ELISA was first used to detect proteins for both filter arrays (66,67) and glass arrays (68). Analyte-obvezujuća i zadržavanje na protein preko polja prihod termodinamički promidžbenom obvezujuća mehanizam sličan je hibridizacija of nucleic acid probe do cilja. No, otkrivanje ciljeva obvezuju da proteini je znatno složeniji od toga DNA microarray detekcija (9). Trenutno različite metode detekcije se ispituje. Na primjer, ELISA je prvi put korišten za detekciju proteina za oba polja filter (66,67) i staklo nizovi (68).  ELISA based detection methods have the disadvantage of non-specificity of protein-antibody interactions, leading to many false positives.   Radioisotope labeling was used by Ge et al. (22), radioisotope labeling to study protein–protein, protein–DNA, protein–drug interactions on filter arrays.  Zhu et al. used radioisotope labeling to conduct kinase assays of different substrates by using purified yeast kinase proteins on a array (36).  The preferred method of detection is fluorescence detection because these methods are generally safe, extremely sensitive, simple and can have very high resolution.  These detection methods are also compatible with standard microarray scanners. ELISA za otkrivanje metode temelji se na nedostatak ne-Specificity of-antitijelo protein interakcije, koje bi dovele do mnogih neistinit određen. Radioisotope označavanje je koristio Ge et al. (22), radioisotope označavanje na studij protein-protein, protein-DNA, protein - droge interakcije na filtar nizovi. Zhu et al. radioisotope koristi za označavanje ponašanja kinaza eseji o raznim podlogama pomoću očistili kvasac kinaza proteina na polje (36). željene metode detekcije je fluorescencija otkrivanje jer ove metode su općenito sigurno, vrlo osjetljivim , Jednostavna i može imati vrlo visoke rezolucije. Otkrivanje Te metode su također kompatibilan sa standardnim microarray skenera. Generally, a chip is either directly probed with a fluorescent molecule (eg a fluorescently labeled protein or small molecule, by using a tagged probe (eg biotin), which can then be detected in a second step using a fluorescently labeled affinity reagent (eg streptavidin) (16,56). Another fluorescent labeling method is rolling circle amplification (RCA), which is also extremely sensitive (32). Općenito, čip je bilo izravno s probed fluorescentnu molekule (npr. fluorescently etiketom protein ili male molekule, pomoću označene ispitne (npr. biotin), koja potom može biti otkrivena u drugom koraku koristeći fluorescently etiketom afinitet reagens (npr. streptavidin ) (16,56). Drugi je fluorescencija označavanje metoda rolling circle pojačanje (RCA), koji je također izuzetno osjetljiva (32).
Although the proteomes under comparison can be labeled in a comparable fashion with fluorophores, the reproducibility of these chemical reactions is poor and interference with the protein-antibody interactions presents an additional complexity (9). Iako je proteomes pod usporedbu mogu biti označene u usporedivim s modnim fluorophores, reproducibilnost od tih kemijskih reakcija je loša i interferencija s-antitijelo protein interakcije predstavlja dodatnu kompleksnost (9). Also, non-uniform labeling of proteins can be addressed by performing a dual-colour ratiometric assay, where an internal standard is present for each target protein which is measured (9). Također, nejednoliko označavanje proteina može se obrati izvođenjem dual-colour ratiometric esej, gdje je jedan interni standard je poklon za svaku ciljnu protein koji se mjeri (9).          A disadvantage of labeling proteins with fluorophores is a reduction of the quantitative accuracy of the assay, as incorporation of the label may alter the binding properties of the proteins (9). Nepogodan za označavanje proteina s fluorophores je smanjenje od kvantitativne točnost, esej, kao trgovačko društvo u oznaku svibanj mijenjati binding properties od proteina (9).

Although direct protein labeling detection methods are still widely used, the intrinsic problems mentioned has resulted in the increasing use of label free detection methods for protein microarrays.  These methods are mass spectrometry (MS), atomic force microscopy (AFM) (70), and surface plasmon resonance (SPR) (71). Iako direktni protein označavanje otkrivanje metode su i dalje naširoko koristi, pravi problemi spominje je rezultirala je povećanjem korištenja label besplatno metode za detekciju proteina microarrays. Te metode su masena spektrometrija (MS), atomic force microscopy (AFM) (70), i surface plasmon resonance (SPR) (71).
Non-labeling methods have advantages as a direct detection approach for antibody microarrays since labeling molecules affects protein activity. Non-označavanje metode imaju prednosti kao direktni pristup za otkrivanje antitijela microarrays od označavanje molekula utječe na protein aktivnosti. SELDI (surface-enhanced laser desorption/ionization) mass spectrometry has been used to detect low-density arrays of captured proteins (69). SELDI (površine poboljšane laserska desorpcija / ionizacija) spektrometrija masa je korištena za otkrivanje niske gustoće nizovi zarobljenih proteina (69). Proteins are captured on a metal surface array (SELDI protein array) and are vapourized using a laser beam.  Analysis using mass spectrometry data is then performed in order to reveal the identities of these proteins. Proteini su zarobljeni na metalne površine array (SELDI protein array) i vapourized su pomoću laser beam. Analiza pomoću mass spectrometry podataka izvršio je zatim kako bi se otkrilo kako je identitet tih proteina.

Atomic force microscopy (AFM) method uses surface topological changes to identify the captured proteins on an antibody array (70). Atomic force microscopy (AFM) metoda koristi površine topološke promjene u identificiranju zarobljeni proteina na antitijela array (70).  When rabbit IgG is immobilized on a gold surface and binds to its complimentary antibodies, goat ant-rabbit IgG, AFM detects the increase in height, and thus is able to measure binding interactions.  However in order to study the kinetics of antigen–antibody interactions, real-time detection methods will be useful. Kada zec IgG je immobilized na površini i zlato povezuje sa svojim pozdravni protutijela, koza ant-zec IgG, AFM otkriva porast u visinu, i tako je u mogućnosti mjeriti obvezujuća interakcije. Međutim, kako bi se studija je kinetika antigen-antitijelo interakcija , Real-time otkrivanje metode će biti korisna.  Surface plasmon resonance (SPR) has matured into a versatile detection tool to study the kinetics of receptor–ligand interactions with a wide range of molecular weights, affinities and binding rates (72-74). Surface plasmon resonance (SPR) ima sazrevali u svestrani alat za otkrivanje studija je kinetika receptor-ligand interakcija sa širokim rasponom molekularne težine, naklonosti i obvezujuća stopa (72-74). Commercial SPR chips are available however their detection resolution is limited.  A sensor surface with 64 individual immobilization sites in a single flow cell was developed (75).  An antibody array biosensor was also developed to study the kinetics of antigen binding using a planar waveguide as the detection method. Poslovni SPR čips dostupni su ipak njihova detekcija rezolucija je ograničena. Senzor površine s 64 pojedinačnih immobilization web-mjesta u jednoj protoka stanica je razvijena (75). Antitijela An array biosenzor je također razvio na studij u kinetika antigen binding koristeći planar kao valovod detekciji metoda. Using this method, the group demonstrated that significant signal intensity could be achieved from spots as small as 200 mm in diameter. Koristeći ovu metodu, grupi pokazalo da značajan signal intenzitet može biti postignut iz malih mjesta kao i 200 mm. It is therefore expected that this approach will be suitable for high-throughput and parallel kinetics studies. To je, dakle, očekuje da ovaj pristup će biti pogodna za visoke propusnosti i paralelno kinetika studija. (76).

Range of Detection Raspon Detection
Another difference between protein and DNA microarrays is that protein concentrations in a single biological sample or cells are several orders of magnitute greater than that for mRNAs.  Thus protein chip detector systems must have a very large range of detection operation – up to a factor of 1014, compared to 104 for mRNA.  Thus an antibody with nanomolar affinity to a particular target will be saturated by the presence of this target at micromolar concentrations and will fail to detect pico- or femtomolar target levels.  Thus accommodating rare and abundant proteins will probably require separate arrays (7,8,9). Još jedna razlika između proteina i DNA microarrays je da koncentracije proteina u jednom biološkog uzorka ili stanice su nekoliko redova magnitute veći od onoga za mRNAs. Tako protein detektor chip sustavi moraju imati vrlo velik raspon otkrivanje operacija - do faktor 1014 , U odnosu na 104 za mRNA. Tako je jedan antitijela s nanomolar afinitet za određenu ciljnu će biti zasićen po prisutnosti taj cilj to micromolar koncentracije i neće uspjeti da se otkriju ili pico-femtomolar target razinama. Tako ljubazan rijetke i bogata proteina vjerojatno će zahtijevati zasebna polja (7,8,9).
Multiple antibodies with varying affinities for the target may be positioned at different areas of the array however studies have shown that only 20% of arrayed antibodies provide measurements of proteins at low concentrations (33). Višestruki protutijela s različitih naklonosti za cilj svibanj biti smješten na različitim područjima u polje međutim studije pokazuju da samo 20% zaodjenu protutijela osigurati mjerenja na niskim koncentracijama proteina (33).

Next: Protein Production for Protein Arrays Next: protein-protein Proizvodnja za Razvrstati

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

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

Types of Antibody and Protein Chips Vrste Antitijelo i protein Chips