Antibody Arrays

Quantitative data gives you the actual concentration (pg/ml, for example) of the analyte, while semi-quantitative data gives you the relative fold-change of the analyte compared to the concentration in another sample.

The main difference between the glass-slide and membrane-based arrays is the type of detection used. Glass-slide-based arrays use fluorescence and need a compatible laser scanner, while membrane-based arrays use chemiluminescence, which can be detected with most western blot imaging systems.

Another difference is the required sample size. Glass-slide assays use smaller sample sizes than membrane-based assays. Glass slides require ~70 ul – 100 ul, while the membranes require at least 1 ml, after dilution.

Lastly, glass-slide-based arrays are generally cheaper than membrane-based arrays.

For our membrane-based arrays, any chemiluminescence imaging system such as an X-ray film developer, CCD camera, or gel documentation system should work. Imaging systems using near-infrared flours, such as the Li-Cor Odyssey and Typhoon systems, also work extremely well with our membrane arrays.

For the glass slide-based arrays, you’ll need a gene microarray laser scanner. A list of specifications and compatible laser scanners can be found here:

Compatible Laser Scanners for Glass Slide Arrays

Most of our arrays are compatible with any biological fluid. This includes cell culture media, cell lysates, tissue lysates, and all clarified body fluids (serum, plasma, urine, cerebrospinal fluid, BAL, saliva, tears, etc.)

There are exceptions for label-based arrays (L-Series), which have limited compatibility with lysates and non-serum/plasma body fluids. Our phosphorylation arrays, which detect membrane-bound receptors, are only validated for use with cell and tissue lysates.

In general, any cell-free extract that contains soluble, non-denatured proteins is likely to work with our arrays.

Our tips for sample preparation can be found here:

Tips on Sample Preparation

The positive control spots are standardized amounts of biotinylated IgG. They are used for signal normalization, monitoring of the detection step, and to help orient the array image.

The negative control spots are printed with buffer only, and thus are not expected to give signals. Negative control spots are used for background subtraction.

The areas on the array map labeled as “blank” are empty (there is nothing printed there). Both the negative control and blank spots should give similar intensity values, and either one may be used to represent the background.

For our membrane arrays, data collection can be accomplished by any densitometry software. CCD camera-based imaging systems are typically equipped with a built-in densitometry software application.

Alternatively, there is a free densitometry program available through the NIH (Image J), which can be downloaded here. More information on how to use Image J can be found here.

For glass slide-based arrays, data extraction is accomplished by the software application associated with the laser scanner. For faster spot identification, we can provide you with a free GAL file which can be downloaded here. More information regarding glass slide array data extraction can be found here.

Normalization is used to compare data between arrays (i.e., different samples) by accounting for the differences in signal intensities of the positive control spots on those arrays. We can do this because the positive control is a controlled amount of biotinylated antibody that is attached to the solid support. The amount of signal from each of those spots is dependent on 1) the amount of the reporter (streptavidin-HRP or streptavidin-fluor) bound to that antibody and 2) the length of exposure time (for chemiluminescent detection).

Since these factors proportionally affect the signal intensity of every spot on the array, the differences in the positive control signals between arrays will accurately reflect the differences between other spots on those arrays.

One array must be defined as the "reference array“ to which the signals of other arrays are normalized. It is up to the researcher to define which array should be the reference.

You can calculate the normalized values as follows:
X(nY) = X(Y) x P1/P(Y)

• P1 = the average signal density of the positive control spots on the reference array
• P(Y) = the average signal density of the positive control spots on Array Y
• X(Y) = the signal density for a particular spot on Array for sample “Y”
• X(nY)= the normalized value for that particular spot "X" on Array for sample “Y"

Please note that RayBiotech’s analysis software tools can perform the normalization automatically. More information about our analysis tools can be found here.

No problem! Simply send your slides to us and we can scan them for you. We will provide you with the scanned image (TIF file) and the densitometry values with full analysis. Learn more about our scanning services here:

Scanning and Data Analysis Services

Different arrays have different sensitivities. You can find more information here:

Quantibody^® Array

Human Cytokine Antibody Array

Mouse Cytokine Antibody Array

We’ve created a helpful guide to help you organize your slides. You can find it here:

Quantibody Standard Options