Antibody Arrays FAQs (Frequently asked questions) are answered by Creative Biolabs to help your project a success.
Antibody array is also known as antibody microarray, it is a novel protein microarray technology. In short, a collection of capture antibodies are spotted and fixed on a solid surface (glass, plastic, membrane, or silicon chip, etc), then the interaction between the antibody and its target can be measured. Antibody arrays are widely used in protein expression analysis from various biofluids such as serum, plasma and cell or tissue lysates. So far, the method has been used for both basic research and medical and diagnostic applications.
Normally, the arrays can be divided into membrane-based arrays and glass slide-based arrays. For membrane-based arrays, a variety of chemiluminescence imaging system is suitable such as an X-ray film developer, CCD camera, or gel documentation system. For imaging systems, the Li-Cor Odyssey and Typhoon systems can meet the membrane-based arrays.
For the glass slide-based arrays, a gene microarray laser scanner is essential.
The main difference between the membrane-based and glass-slide arrays is the type of detection used. Membrane-based arrays usually use chemiluminescence, which can be detected with most western blot imaging systems. Glass-slide based arrays often use fluorescence and need a compatible laser scanner. In addition, the sample size needed for the glass-slide based is smaller than the membrane-based. The membranes need at least 1 mL, while glass-slides only require about 70 uL-100 uL. Lastly, the price for the glass-slide based arrays is cheaper generally.
Generally, most of samples can be detect using the assays including cell culture media, cell lysates, tissue lysates, and all clarified body fluids such as serum, plasma, urine, cerebrospinal fluid, BAL, saliva, tears and so on.
For different samples, the sample preparation processes are different:
Serum-free or low-serum medium samples are preferential due to serum contain cytokines that may significantly cause non-specific background signals. If it is necessary to test serum containing medium, we recommend running an uncultured media blank to assess baseline signals.
(1) On day 0, seed approximately 1×106 cells in 100 mm tissue culture plate with complete medium.
(2) On day 3, remove and replace medium with 6-8 mL of serum-free or low serum containing medium.
(3) On day 5, collect medium into 15 mL tube. Centrifuge at 2,000 rpm, 4ºC for 10 minutes. Save the supernatant. Transfer the supernatant into 1.5 ml centrifuge tubes. Store supernatant at -80ºC until experiment.
(1) Collect whole blood into a tube with EDTA, Citrate or Sodium heparin.
(2) Centrifuge 10 minutes at 3,000 rpm.
(3) Aliquot into small tubes and store at -80°C until use.
(1) Collect whole blood into a tube without any additives.
(2) Keep 20 minutes at room temperature.
(3) Centrifuge 10 minutes at 3,000 rpm.
(4) Aliquot into small tubes and store at -80°C until use.
(1) Collect urine without adding stabilizers.
(2) Centrifuge the samples hard (eg. 10,000 x g for 1 min or 5,000 x g for 2 min).
(3) Aliquot, quick freeze in dry ice/methanol bath, and store at -80°C until use.
Cell or tissue lysates can be prepared using conventional methods, namely, homogenization of cell or tissue in lysis buffer, such as RIPA or other formulations optimized for immunoprecipitation.
The positive control spots are used for signal normalization, monitoring of the detection step, and to help orient the array image. Positive controls often are standardized amounts of biotinylated IgG.
The negative control spots are printed with buffer only, and no signals can be obtained. Thus, negative control spots are used for background subtraction. Besides, the blank spots on the array map labeled as “blank” are empty, which are nothing printed there. Both the negative control and blank spots should present similar intensity values, and either one may be used to represent the background.
If you still have other questions about antibody arrays, please feel free to contact us.
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