Q: What are the major applications of the antibody microarray?
A: The antibody arrays are designed for qualitative protein expression or phosphorylation profiling, screening, and comparison between normal, diseased or treated samples.
Q: How many slides are included in one set of arrays?
A: There are two identical slides included in each set of arrays. Each slide can be used to analyze one sample. You can analyze two samples with each set of arrays.
Q: What samples can be used for analysis?
A: Proteins from cell extracts, tissue lysates, serum samples, or culture media can be used for analysis.
Q: Do I need to run a control/known sample?
A: It is recommended to run a control or known sample along with the test samples.
Q. How many cells are needed?
A: The amount of protein present in cells may vary with cell type. We typically use 1 to 10 million cells. Start with 3-5 million cells whenever possible.
Q: What scanners can be used for detection?
A: Any fluorescence based scanner, that is compatible with 3 in x 1 in (76 mm x 25 mm) microscope slides, can be used for detection. Here is a list of commonly used compatible and incompatible detection systems. If you do not have access to a scanner, please consider our array scanning and image analysis service.
Q: What does it mean when an antibody has a name like this, p53 (Ab-15) or p53(Phospho-Ser15)?
A: The number indicates phosphorylation site. For example, antibody p53 (Ab-15) is made from a synthetic nonphosphopeptide derived from human p53 around the phosphorylation site of Serine 15. It detects endogenous levels of total p53 protein. Antibody p53(Phospho-Ser15) is made from a synthetic phosphopeptide derived from human p53 around the phosphorylation site of Serine 15. It detects endogenous levels of p53 only when phosphorylated at Serine 15.
Q: In the phospho-antibody arrays, why are there multiple antibodies for a single protein?
A: These are highly specific antibodies made to recognize the residues around specific phosphorylation sites on the same protein. For example, c-Jun (Phospho-Thr91) detects endogenous levels of c-Jun only when phosphorylated at Threonine 91, and c-Jun (Ab-91) detects total c-Jun protein by recognizing specific residues around phosphorylation site threonine 91; c-Jun (Phospho-Tyr170) detected endogenous levels of c-Jun only when phosphorylated at Tyrosine 170, and c-Jun (Ab-170) detects total c-Jun protein by recognizing specific residues around phosphorylation site tyrosine 170.
Q. How many cells are needed to obtain 100 micrograms of protein?
A: The amount of protein present in cells may vary with cell type. We typically use 1 to 5 million cells to get 200 – 400ug of protein.
Q: Can I use other types of lysis and/or extraction buffer instead of the Extraction buffer provided in the Array Assay Kit?
A: Yes, it is possible to use other lysis buffers to lyse cells and tissues; however, the buffer must be free of Tris. The presence of Tris in cell lysates or extracted protein sample can adversely affect biotinylation of protein samples. For instance, the RIPA Lysis and Extraction buffer from Pierce Biotechnology contains Tris. If this buffer was used to extract proteins from cells, please be sure to remove the buffer from your protein extract and replace with the Labeling Buffer provided in the Antibody Array Assay Kit before proceeding to the next step. We recommend the following columns for buffer exchange (removing Tris): Spin columns included in the Antibody Array Assay Kit; Millipore, Microcon YM-10 filters (Catalog: 42406); Sephadex G-25 columns.
Q: I need to use microbeads for cell separation and purification. Will the beads interfere with the antibody arrays?
A: In generally, microbeads used for separation and purification do not affect antibody array assays.
Q: Can I add protease inhibitors to the lysates?
A: The reagents provided in the Antibody Array Assay Kit do not contain protease or phosphatase inhibitors. To prevent protein degradation, once you start the extraction, you should work quickly and proceed diligently towards the array analysis step. Alternatively, you may use inhibitors if you prefer or plan to store the proteins for a week or longer.
Q. What is the amount of proteins needed for coupling?
A: Typically 60-100ug of total protein is used for biotin labeling and coupling.
Q: How important is it to rinse the slides extensively with DI water after blocking and coupling?
A: It is extremely important to rinse the slide extensively with DI water. Any residual reagents on the slide surface may cause non-uniform background. Rinsing the slides with DI water extensively helps achieve better background uniformity.
Q: Can I used a different dye-streptavidin instead of Cy3-Streptavidin for detection?
A: Yes. Cy3-Streptavidin is not the only option. The choice of dye depends on your scanner’s specifications. For commonly used 532 nm and 635 nm lasers, detection reagents include Cy3 or Cy5-Streptavidin, or Alexa Fluor 555 or 647 conjugated streptavidin.
Q. What are the key steps in getting good results?
A: The first key step in getting good results is protein extraction. Make sure to vortex the beads with cells/tissues for 30 seconds every 10 minutes for 1 hours. This process will fully disrupt the cells/tissues and release protein. If you are working with tissues, be sure to remove as much blood as possible. Blood left in the sample can cause high background and non-specific binding. Secondly, it is very important that the cell lysate supernatant is very clear. Impurities in the supernatant (dirty lysate) can cause low labeling efficiency and non-specific binding. Be sure to only use the top, clear layer of the lysate supernatant for labeling. The supernatant should be as clear and transparent as water. If the supernatant still appears cloudy (unclear) at the end of the extraction protocol, freeze it at -70 for 10min, then spin at 10000xg. Save the clear layer of the supernatant.
Another important step is the rinses with DI water. It’s absolutely crucial to wash the slides EXTENSIVELY with DI water after blocking, coupling and detection. Increase the number of washes if needed. Agitate the water during wash. It will help remove any residual reagent from the slide surface. The video below shows washing the slides with DI water using a 50-ml conical tube. If you are handling multiple slides at once, you can wash the slides together using a slide rack (watch video).
Q: What scanners can be used for detection?
A: Any fluorescence based scanner, that is compatible with 3 in x 1 in (76 mm x 25 mm) microscope slides, can be used for detection. Here is a list of commonly used compatible and incompatible detection systems. If you do not have access to a scanner, please consider our array scanning and image analysis service.
Q: How long can the finished arrays be stored before they are read on a scanner?
A: The arrays should be scanned as soon as possible after the experiment is complete. If you do not have access to a scanner immediately, store the finished arrays in a non-transparent box (to protect them from light) at room temperature for two or three days before they can be read on a scanner.
Q: Does Full Moon BioSystems provide a software for image analysis/data extraction?
A: We do not provide an image analysis software. Any image quantification software program may be used for image analysis. We do provide a GAL file (GenePix Array List) for each array, which may be used to generate quantification grids for image analysis. To learn more about GAL files including explanations of the data records, visit GenePix Array List Format on mdc.custhelp.com/.
Q: I’m getting an error when loading the GAL file.
A: First, make sure your analysis software supports GAL files. Second, check the scan resolution and image scaling factor. Our GAL files are created for images scanned at 10 micron resolution without any scaling factor. For images scanned at different resolutions and/or with scaling factors applied, modify the parameters accordingly.
Q: My image analysis software does not support GAL files. How do I create an analysis grid?
A: If your image analysis software is not compatible with the our GAL files we provide, you will need to create a new analysis grid. General array parameters including spot diameter, spot-to-spot distance, and block-to-block distance are necessary to create a grid. For array specific information including, antibody ID, name, and total number of blocks, refer to the array map of each array, which is available under the “Documentation” tab on each array’s web page. Please refer to your analysis software user’s guide for detailed instructions.
Q: My Western blot results show a 10-fold change between my control sample and my treated sample, but the array results show much smaller change. Is this normal?
A: In general, the fold change from the array results are much smaller in value than the Western blot results. However, the trends are usually the same.
Q: My Western blot results indicate a large change between my two samples, but the array results show no change. What are the possible explanations?
A: The antibodies in the phospho arrays recognize specific residues around the phosphorylation sites. The phospho antibodies detect the protein only when the protein is phosphorylated. The non-phospho antibodies detect total protein. The proteins used in the antibody array assays are not denatured, so their 3D folding structures are retained. Therefore, they are recognized by the antibodies only when the specific residues are exposed and available for binding. If certain proteins in the sample are not detected, it is usually because the binding residues were not accessible. In other cases, a protein can form a complex with another protein immediately after phosphorylation. This phenomenon can block the phosphorylation site from being recognized by antibodies; as a result, the protein is not detected.