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Antibody Array FAQs
- What is the difference between quantitative and semi-quantitative?
- What is the difference between the membrane-based and glass slide-based arrays?
- What equipment do I need to process the arrays?
- What types of samples are compatible with the arrays?
- How do I prepare my samples?
- What are the positive and negative controls on the arrays?
- How do I extract my data from the arrays?
- How do I normalize my array data?
- What can I do if I want to use the glass slide arrays but don’t have a laser scanner?
- What is the sensitivity of the arrays?
What is the difference between quantitative and semi-quantitative?
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 in two or more samples being compared.
What is the difference between membrane-based and glass slide-based arrays?
There are a few differences between the glass-slide and membrane-based arrays. The main difference is the type of detection used. Our glass-slide based arrays use fluorescence and need a compatible laser scanner (see link below the next answer for a list of compatible/recommended laser scanners); while our membrane-based arrays use chemiluminescence, which can be detected with most western blot imaging systems.
The sample size needed for the glass-slide based is smaller than the membrane-based. Glass-slides require ~70 ul – 100 ul, while the membranes require at least 1 ml, after dilution.
Lastly, the price for the glass-slide based arrays are generally cheaper.
What equipment do I need to process the 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, a gene microarray laser scanner is needed. A list of specifications and compatible laser scanners can be found here:
What types of samples are compatible with the 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.)
The exceptions are the 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.
How do I prepare my samples?
Our tips for sample preparation can be found here:
What are the positive and negative controls on the arrays?
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.
How do I extract my data from the arrays?
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: http://rsb.info.nih.gov/ij/ More information on how to use Image J can be found here: http://imagejdocu.tudor.lu/imagej-documentation-wiki.
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.
How do I normalize my array data?
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. To learn more about our analysis tools, click here.
What can I do if I want to use the glass slide arrays but don’t have a laser scanner?
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:
What is the sensitivity of the arrays?