Key Product Features:

• Protein binding: high binding nitrocellulose membrane pads

• signal: high signal to noise ratio (> 50) at very low protein concentrations (< 0,5 ng)

• background: very low native background in fluorescence

• compatibility: with all spotters and scanners

• formats: different nitrocellulose pad formats to fit incubation chambers

• assays: recommended for forward and reverse phase pProtein arrays (microarrays or protein biochips) have become an indispensable tool for many proteomic applications as well as for new multi parameter clinical diagnostic tests. The multiplicity of protein spots, displayed in an intricate pattern on the slide surface, allow looking at various interactions simultaneously. Therefore, protein arrays, used for example in drug or antibody cross reactivity screening or in protein expression pathways in tumor cells, considerably accelerate the quest for new drug targets and disease markers.

Modified glass surface has become the most widely used substrate for nucleic DNA arrays. However, proteins are quite different from nucleic acids. They are heterogeneous with limited stability while DNAs are uniform and stable. Proteins easily lose their activity through denaturation and dehydratation as DNAs keep their activity even when denaturated. Protein chemistry is not easy with limited amplification method whereas for DNA restriction enzymes and PCR exist. All these differences explain why proteins need a different solid substrate for arrays. Today the best and most known surface for protein arrays is a nitrocellulose membrane.

After several years of development, a new manufacturing process has been designed. This new validated robotic equipment allows manufacturing the new UniSart 3D slides with an unmatched consistency.

The new production process brings the following advantages:

• Tightest thickness specification with |- 1 µm maximum variation. A constant thickness will allow for a homogeneous membrane structure and thus a uniform protein binding and spot morphology.

• Consistent and larger batches. The new process enables the production of nitrocellulose coated slides on a continuous mode. This offers the guaranty for better intra and inter lot consistency

The UniSart® 3D slide is a special glass slide coated with a thin microporous nitrocellulose membrane. The pad on the slide is a 3-dimensional microporous nitrocellulose membrane, which has been optimized by Sartorius Stedim Biotech to show high protein affinity and binding capacity in all microarray application.

Glass slide : The glass used for the UniSart® slide is a high quality borosilicate glass slide. It has been further optimized to promote a perfect attachment of the nitrocellulose coating. When the pad format permits it, a 128 barcode label can be included on each slide for ease of identification and reliable tracking. Slide dimensions are the industrial standard in order to fit with existing instruments used by the microarray community.

Nitrocellulose membrane pad : Due to its strong affinity for proteins, nitrocellulose membranes have been used for decades in many applications like blotting and lateral flow immunoassays. Hence, nitrocellulose membranes have become the reference substrate for protein binding. The unique surface characteristics of these membranes enable an immediate binding of proteins through electrostatic and hydrophobic interactions. This binding is not reversible under standard conditions and stabilizes proteins in their functional natural activity state. Because the 3-dimensional microporous structure offers a large internal surface area, a much bigger amount of proteins can be fixed compared to standard 2-dimensional surfaces like glass. The 3D nitrocellulose membrane covering the UniSart slides has been further optimized to show:

• An increased signal to noise ratio, even at very low protein concentrations

• A very low background of the native array slide

• A perfect spot morphology

Application :

UniSart® 3D slide has been designed to be the best substrate for protein array applications like:

• Forward (direct) protein array

• Reverse phase protein array

Forward protein array : Forward protein array ,also called capture arrays, are slides having tiny amounts ( in picoliter range) of purified known proteins spotted in an array format. More than 15 000 active spots can be printed. Following exposure with a defined sample, a simultaneous reaction of the various components of the sample can take place with all the individual spotted molecules. Specific antibodies are commonly used as capture molecules but antigens can be spotted directly as well to detect auto-antibodies in serum, for example. Peptides, aptamers, nucleic acids and enzymes may all act as alternative capture molecules. Taken together, a wide variety of protein–protein as well as protein-alternative binding partner interactions can be investigated on a protein microarray format. This interaction is then visualized and quantified with the use of classical immunostaining tools including enzyme-coupled or fluorescent labeled antibody

Reverse Phase Protein Array : In this assay format, a multiple micro volume of cell lysate, plasma or body fluids are spotted onto the slide. The reverse phase array is then incubated against one single specific marker, usually a high quality validated antibody. This protein array is designed as micro-scale dot-blot platform that allows for quantitative measurement of protein expression levels and/or post-translational modification in a large number of biological samples simultaneously. One reverse phase slide can accommodate several hundreds to thousands of samples that are printed in series of replicates. Detection is performed using specific combinations of primary and secondary antibodies. Because of the small amount of protein available, a signal amplification step is commonly introduced. The intensity of the generated chemiluminescent, fluorescent or colorimetric signals is then quantified.

Models

One pad slide :

Description : UniSart® 3D slide with one nitrocellulose membrane pad of 51 mm on 21 mm or 60 mm on 21 mm and a bar code label, box of 25 slides

Catalogue Number

One pad 51 mm on 21 mm ref.: 2UNY2GW060021M1G

One pad 60 mm on 21 mm ref.: 2UNY2GW051021M1G

2-pad slide :

Description : UniSart® 3D slide with two nitrocellulose membrane pads of 20 mm on 20 mm and one bar code label, box of 25 slides

Catalogue Number : 2UNY2GW020020M2G

16-pad slide :

Description : UniSart® 3D slide with 16 nitrocellulose membrane pads of 6 mm on 6 mm, box of 25 slides

Catalogue Number : 2UNY2GW00600616G

UniSart® 3D Slide with Custom Pad

Different custom pads can be easily produced with the new manufacturing process. Pads can be of any size within the limit of the standard glass slide or within the 96 plate size.

Ask us for prototypes and a quote for minimum quantities

In-depth

Standard protocols:

Preliminary remarks: UniSart® 3D slides can be used with and without an incubation chamber. All standard incubation chambers already available are compatible with the UniSart® 3D slides.

Autostainer equipment can be used for incubation and washing steps of the slide. During longer incubation period, special care should be taken to avoid drying of the slide. The installation of a small humidifier inside the equipment and/or positioning of a hydrophobic ring around the pads through available pens or seals are recommended measures to prevent drying of the slides

Forward protein array:

Every assay is variable and is highly dependent on the specificity of the antibodies used. The protocol here included should be taken as a general guideline of the steps involved.

Antibody sandwich assay:

• Antibody (10-500 µg/ml) is diluted in spotting buffer (e.g.PBS, Trehalose 0.5%)

• Spotting is performed with a glass nozzle (non-contact)

• Fixation of proteins by drying 15 min at 37°C

• Blocking 1 hr at RT° with gentle shaking in PBS-0,1% Tween (PBS-T) and 2%BSA. Initial wetting of the whole nitrocellulose surface should be done carefully. Attention also needs to be given so that the slide remains wet throughout all steps.

• Incubation with biological sample in a buffer containing protease inhibitors ( 2 hrs/RT° - OV 4°C).

• 3 x15 min wash in incubation buffer.

• Incubation with primary antibody OV at 4°C in PBS-T/2% BSA.

• 3 x 15 min wash in PBS-T.

• Incubation with fluorescent-labeled secondary antibody 2 hrs at RT° in PBS-T

• 3 x 15 min wash in PBS-T

• Short dip in distilled water

• Drying of slide and scan for signal intensity quantification

Reverse phase protein array :

Similar to “Forward protein array”, each assay is unique and the specific conditions of a given system should be tested individually. Here described, is one classical example.

• Biological samples are lysed in lysis buffer (e.g. Laemmli buffer) containing protease inhibitors.

• Samples (1 mg/ml) are spotted with contact spotter (e.g. solid pin) and dried overnight.

• Because an amplification signal step is essential for reverse phase application, specific blocking steps (e.g. avidin/biotin block, peroxidase block) have to be performed in addition to protein block.

• Slides are then incubated in Tris buffer/Tween 0,1% (TBS-T), 5%BSA for protein block.

• Incubation with primary antibody OV at 4°C in TBS-T, 5% BSA.

• 3 x 15 min wash in TBS-T.

• Incubation with secondary antibody 1 hr at RT° in TBS-T, 5% BSA

• 3 x 15 min wash in TBS-T

• Amplification steps according to the system used (e.g. tyramide amplification) including generous wash steps

• Short dip in distilled water

• Drying of slide and scan for signal intensity quantification