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Protein Attachment to Microarray Chips Protein Tillæg til microarray Chips

Protein and Antibody Microarrays Protein og Antibody Microarrays

Creation and Manufacture of Protein and Antibody Microarray Chips Creation og fremstilling af protein og Antibody microarray Chips

Attachment Vedhæftet
To attach proteins to a solid surface, the surface of the substrate has to be modified to achieve maximum binding capacity (8,27).  The proteins are attached to the chip on a protein attachment layer (see Figure 3).  This layer is typically an organic film which varies with the nature of the application.  A variety of materials have been studied including agarose (39), dextran-based hydrogel (40), porous polyacrylamide hydrogel  hydrophilic polymers and polyamino acids (41). At knytte proteiner til en fast overflade overfladen af substrat der skal ændres for at opnå maksimal bindende kapacitet (8,27). Proteiner er knyttet til chip på et protein udlæg lag (se figur 3). Dette lag er typisk en organisk film, der varierer efter karakteren af ansøgningen. Forskellige materialer har været undersøgt, herunder agarose (39), dextran-baserede hydrogel (40), porøse polyacrylamid hydrogel hydrofile polymerer og polyamino syrer (41).
A convenient attachment method used nitrocellulose-membrane or poly-L-Lysine coated glass such that proteins could be passively absorbed onto the surface through non-specific interactions (34,42,43).  The attached proteins bind onto the surface in random orientations and can be washed off under stringent washing conditions.  However, the noise level is usually higher because of the non-specific absorption/adsorption. En praktisk udlæg anvendte metode nitrocellulose-membran eller poly-L-lysin-coatede glas sådan at proteiner kan være passivt absorberet på overfladen gennem ikke-specifikke interaktion (34,42,43). Vedlagte proteiner binde på overfladen i vilkårlig retningslinjer og kan vaskes ud under strenge vask betingelser. Men støjniveauet er normalt højere på grund af den ikke-specifikke absorption / adsorption.
A more specific and stronger attachment is achieved by creating reactive surfaces on glass that can covalently cross-link to proteins (31,36,26).  A bifunctional silane cross-linker is used to form a self-assembled monolayer (SAM), which has one functional group that reacts with the hydroxyl groups on glass the glass surface, and another group which is free to react with primary amine groups of proteins or can be further chemically modified to reach maximum specificity (44,45).  Another variation is gold-coated glass (46,47).  The advantage of gold-coated chips is that SPR and mass spectrometry can be integrated as detection methods to monitor the dynamics of the reaction, and to identify the captured molecules. En mere specifik og stærkere vedhæftede fil er opnået ved at skabe reaktive overflader af glas, der kan covalently cross-link til proteiner (31,36,26). En bifunctional silan cross-linker bruges til at danne en selvstændig samles monolayer (SAM), som har en funktionel gruppe, der reagerer med hydroxyl grupper på glas glas overflade, og en anden gruppe, som er gratis at reagere med primær amin grupper af proteiner eller kan blive yderligere kemisk modificerede at nå maksimal specificitet (44,45). En anden variation er guld - coatede glas (46,47). Fordelen af guld-belagt chips er, at SPR og mass spectrometry kan integreres som detekteringsmetoder til at overvåge dynamikken i den reaktion, og at kortlægge de fangede molekyler.
The above-mentioned covalent cross-linking approaches however have a disadvantage. Ovennævnte kovalente tvaerbinding tilgange dog have en ulempe. Due to the fact that reactive ligands also exist in the side chains of proteins it is possible that their random attachment may alter the native confirmation of proteins, reduce the activity of proteins, or make them inaccessible to probes (8,27). Da reaktiv ligands også findes i den side kæder af proteiner, er det muligt at deres tilfældige vedhæftet fil kan ændre native bekræftelse af proteiner, reducere aktiviteten af proteiner, eller gøre dem utilgængelige for sonder (8,27).
In order to orient proteins uniformily away from the surface of the chip, proteins may be fused with a high-affinity tag at their amino or carboxy termini.  With this method, immobilized proteins/antibodies are more likely to remain in their native conformation, thus allowing the analytes efficient access to the active sites of the proteins.  This method was first successfully demonstrated with the attachment of 5800 fusion proteins containing a His tag onto a nickel-coated glass slide (26).  Other affinity methods such as glutathione/GST have also been used (48). For at orientere proteiner uniformily væk fra overfladen af den chip, proteiner kan være smeltet med en høj affinitet tag på deres amino eller carboxy endestationer. Med denne metode, ubevaegeligt proteiner / antistoffer er mere tilbøjelige til at forblive i deres kropsbygning, og dermed gør det muligt at analysander effektiv adgang til de aktive sites af proteiner. Denne metode blev først succes demonstreret med udlæg i 5800 fusion proteiner indeholder et Hans-tag på en nikkel-belagt glas-slide (26). Andre affinitet metoder såsom glutathion / gst har også blevet brugt (48).
Streptavidin based immobilization methods have been also widely employed to attach any biotinylated biological element to the array surface (49). Streptavidin baseret immobilisering metoder er også i vid udstrækning anvendes til at vedlægge alle biotinylerede biologiske element i array overflade (49).
The chip support material is important because proteins are highly sensitive to physiochemical properties.  For example, polar arrays are chemically treated to bind to hydrophilic proteins however such surfaces are unsuitable for cell membrane proteins (eg G-protein coupled receptors) as they possess hydrophobic domains (9). Chippen støtte materiale er vigtigt, fordi proteiner er meget følsomme over for fysisk-kemiske egenskaber, f.eks. Polar arrays er kemisk behandlet for at binde sig til hydrofile proteiner Men den slags overflader er uegnet til celle membran proteiner (f.eks G-protein koblede receptorer) som de har hydrofobe domæner (9).
Proteins do not behave like nucleic acids, and different proteins will behave in different ways when exposed to the same surface chemistry.  Different types a surface chemistries will thus promote the retention of some proteins and cause denaturation or loss of activity of others.  Therefore, the proper choice of surface chemistry is important as this will allow immobilized proteins of diverse types to retain their secondary and tertiary structures, and thus their biological activity.  This problem is magnified when the number of different spots on the chip increases, as there may be 100 different ways to immobilize 100 different proteins in order to obtain proper folding and function of all the proteins.  Also a significant problem is because the functions of most proteins are currently unknown, so there is no method to actually test whether they are still functional on the chip (7). Proteiner, der ikke opfører sig som nukleinsyrer, og forskellige proteiner vil optræde på forskellige måder, når det udsættes for den samme overflade kemi. Forskellige typer en overflade chemistries vil således fremme lagring af visse proteiner og forårsage denaturering eller tab af aktivitet på andre. Derfor er korrekt valg af overflade-kemi er vigtigt, da dette vil gøre det muligt ubevaegeligt proteiner af forskellige typer at bevare deres sekundære og tertiære strukturer, og dermed deres biologiske aktivitet. Dette problem forstærkes, når antallet af forskellige pletter på chippen øges, da der kan være 100 forskellige måder at immobilisere 100 forskellige proteiner for at opnå korrekt foldning og funktion af alle de proteiner. også et væsentligt problem er, fordi de funktioner fleste proteiner er i øjeblikket ukendte, så der er ingen metode til rent faktisk at teste, om de stadig er funktionelle på chip (7).

Next: Protein Chip Delivery Methods Næste: Protein Chip Delivery Methods

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

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Introduction and Background to Protein Chips and Antibody Chips. Indledning og baggrund for protein-chips og Antibody Chips.

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