Microarray printing

Place a microarray slide in a hybridization chamber and pipette 25 Ail of the labeled probe mixture on the slide surface near one end of the microarray print area. 

Solid pins are also used for microarray printing. These types of pins were adopted from gridding applications where they were primarily used... 

A new quill pin design known as the "trench pen," has been created for high density microarray printing by Stephen Quake s group at the California... 

In this study, the effects of ionic strength and carrier protein (BSA) were examined in terms of the outcome for microarray printing. As noted previously, many factors can unduly influence printing performance. Proteins are notoriously bad when it comes to nonspecific adsorption. It should not be much of a surprise that some portion of a protein probe will adsorb to the printing device, whether it is a stainless steel quill or glass capillary. 

Martinsky, R.S., Microarray printing device including printing pins with flat tips and exterior channel and method of manufacture, US Patent 6,101,946, issued 2000. 

Kirkwood, J., Al-Khaldi, S. F., Mossoba, M. M., Sedman, J., and Ismail, A. A. (2004). Fourier transform infrared bacteria identification with the use of a focal-plane-array detector and microarray printing. Appl. Spectrosc. 58,1364-1368. 

In order to assess the quality of the p53 protein microarrays printed on SAM2 membranes, two different assays are carried out on replica arrays. The antibodybinding assay comprises binding a Cy3-labeled anti-His antibody to the arrayed p53 proteins. This assay is independent of protein activity but provides a measurement of the relative amounts of protein immobilized in each spot. 

Many instruments for contact or non-contact microarray printing are now commercially available. We have been using the Micro Array 03A (Wittech Co., Ltd.) to print our microarrays. This machine uses flat-tip gold-coated needles the needle tip diameter used for our experiments ranges from 100 pm to 500 pm. 

In our experience, the best support for cell-binding assays is a plastic slide. PVDF membrane has high adsorption capacity, and microarrays printed on such membrane are suitable for enzyme-linked colorimetric and phosphorylation assays. However, researchers are encouraged to try all of them for their specific applications. 

The microarray manufacturing method that enables microarray printing without direct contact to the surface is termed non-contact printing. Piezoelectric, bubble-generated, and microsolenoid driven pipettes as shown in Fig. 3 work with the same physical principle as ink-jet printers and are capa-... 

Wu P, Hogrebe P, Grainger DW (2006) DNA and protein microarray printing on silicon nitride waveguide surfaces. Biosens Bioelectron 21 1252-1263... 

Peptide microarrays are prepared by immobilizing many peptide molecules on the surface of a solid support in a small area in an addressable fashion. The immobilization can be achieved via in situ synthesis or chemical ligation through a covalent bond. A hydrophihc linker between the sohd surface and the peptide usually is added to minimize steric hinderance caused by the sohd support. The most commonly used solid support for microarray printing is a standard microscope glass slide. Other solid supports also have been used such as polystyrene, nitrocellulose membranes, PVDF membranes, Hybond ECL membranes, gold surfaces, and chemical vapor deposited diamond films. 

The methods described herein encompass (1) preparation of a whole cell lysate from either cell culture or tissue samples, (2) protein lysate microarray printing, (3) immunostaining, and (4) microarray spot analysis. 

Microarray printing device (Aushon Biosystems 2470 Protein Arrayer, Burlington, MA, USA). 

Prepare lysates within 1 week of microarray printing. Long-term storage (>6 months) of protein lysates may result in protein degradation or diminished protein yield. If needed, store protein lysates at -80 °C or in the vapor phase of liquid nitrogen. 

The limited volume of the total cellular material from biopsy samples makes it necessary to use a slight modification of the lysis buffer recipe as compared with the cell culture lysis buffer. The following protocol describes a method for preparing whole cell lysates from LCM cells. The limited biopsy sample generally precludes the use of total protein assays before microarray printing. To compensate for this, one microarray slide is stained for total protein using a Sypro Ruby Protein blot stain (see Subheading 3.3.3.). 

The limited sample material from microdissected biopsy lysates precludes the use of spectrophotometeric analysis of total protein prior to microarray printing. A microarray slide stained for total protein serves as a tool for normalizing spot intensity between samples (see Note 15). 

Microarray analysis provides a technology platform for massive, parallel analysis of protein-protein interactions (MacBeath and Schrieber, 2000 Joos et al 2002). The difficulty with protein microarray is that proteins do not behave as uniformly as nucleic acids. Protein function is dependent on a precise and fragile 3D structure that may be difficult to maintain in a microarray format A practical challenge posed by proteins derives from their relatively delicate tertiary stracture, which is susceptible to unfolding during microarray printing. In addition, the efficiency and specificity of protein-protein interactions are not nearly as standardized as nucleic acid hybridization. The use of lectin microarrays in glycoform analysis also encounters the similar problems. 

For cDNA arrays, the effects of spatial artifacts and print tips are often accounted for using statistical models. Loess normalization can be performed separately for the genes in each technical block, microarray print tip (print-tip normalization), and sometimes even with different scaling factors (scaled print-tip normalization) (Yang et al., 2002). 

The more commonly used approach for microarray fabrication is the deposition of pre-synthesized molecules by a microarray printer onto substrate, the so-called contact and nmi-contact microarray printing (Fig. 3a, b). Contact arrayers use different-shaped steel pins for direct deposition of droplets onto the substrate by touching the surface with the pins. Nrai-contact arrayers are similar to commercial piezo-driven ink-jet printers and deposit a droplet onto the substrate without coming into contact with the substrate surface. Microarrayers which use other technical approaches are also commercially available and will also be discussed. 

D. Pinkel, D.G. Albertson, G. Hamilton, N.W. Brown, and R. Nord-meyer. Capillary pins for high-efficiency microarray printing device, US Patent 8 283 181, assigned to The Regents of the University of California (Oakland, CA), October 9, 2012. 

Lab-on-Chip Devices for Protein Anaiysis, Rgure 3 A general schematic for microarray printing where the pin head carrying the printing device (pins) Is mounted on a robot arm that carries the sample from the reservoir to the plate (substrate) where it is deposited in an array format (adapted from [2])... 

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