Q: What are the major applications of the antibody microarray?

A: The antibody arrays are designed for qualitative profiling of protein expression, screening, and comparison between normal, diseased or treated samples.

Q: How many slides are included in one set of arrays?

A: There are two slides included in each set of arrays.  There is one array on each slide for analyzing one sample.  You can analyze two samples with each set of arrays.

Q: What samples can be used for analysis and detection?

A: Proteins from cell extracts, tissue lysates, or serum samples 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: What does it mean when an antibody has a name like this, p53(ab-15) or p53(phospho-Ser15)?

A: The number in parenthesis in an antibody's name indicates the site of phosphorylation . For example, p53(Ab-15) was made from a synthetic peptide (non-phosphorylated) derived from human p53 around the phosphorylation site of Serine 15. It detects endogenous levels of total p53 protein.  p53(phospho-Ser15) means that the antibody was derived from a synthetic phosphopeptide derived from human p53 around the phosphorylation site of Serine 315. It detects endogenous levels of p53 only when phosphorylated at Serine 315.  Both phospho-specific antibodies and their non-phospho pairs are included in the array.

Q: In the phospho-antibody arrays, why are there multiple antibodies for a single protein?

A: They are highly specific antibodies made to recognize the different phosphorylation sites on the same protein.  For example, c-Jun(Phospho-Thr91) detects endogenous levels of c-Jun only when phosphorylated at Threonine 91; c-Jun(Phospho-Tyr170) detected endogenous levels of c-Jun only when phosphorylated at Tyrosine 170.

Q. What are the key steps in getting good results?

A: The first key step in getting good results is protein extraction. First, make sure to vortex the beads with cells for 30 seconds every 10 minutes for 1 hours. This process will fully disrupt the cells/tissues and release protein. Secondly, it is very important that the cell lysate supernatant is very clear.  Impurity 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.  Here is a short video showing how we wash the slides with DI water. http://www.youtube.com/watch?v=mst7vnahpP8 If you don't use slide racks, you can submerge the slide under water and then move it back and forth.

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 10 million cells to get approximately 1mg of protein; only 10uL of which containing less than 100ug of protein is used for labeling.  Start with 5 million cells whenever possible.

Q: Can I use other types of cell 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; 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: Can I add protease inhibitors to the lysates ?

A: The reagents provided in the Antibody Array Assay Kit do not contain protease 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 minimum amount of proteins needed for coupling?

A: Typically 40-100ug of total protein is used for biotin labeling and coupling.

Q. Which method is better for drying the slide?

A: For drying the slide after detection, the goal is to remove water from surface as quickly as possible. Compressed nitrogen works better than centrifugation. It's faster and more efficient. Before using nitrogen, hold the slide with your fingers and shake off excess water as much as you can. Then, point the nitrogen nozzle at a 30 degree angle about 20mm away from the slide surface, use the air to push the water off the surface quickly without touching the slide surface. Be sure to keep the air pressure between 20-30psi.

Q: Do I have to use Cy3-Streptavidin for detection?

A: No, you do not have to use Cy3-Streptavidin.  As alternatives, you can use Cy5-Streptavidin, or Alexa Fluor 532 or 647 conjugated streptavidin.

Q: How important is it to rinse the slides extensively with DI water after the slides are subject to blocking buffer and/or wash buffer?

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: How long can the finished arrays be stored before they are read on a scanner?

A: The arrays should be read (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: 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. Click here for a list of compatible and incompatible systems.  If you do not have access to a scanner, we will be happy to scan the arrays for you and provide raw data in Excel format.  Click here for more information on array scanning and image analysis.

Q: Do you provide a software for image analysis?

A: We do not provide an image analysis software. Any image analysis software that came with the scanners or any type of spot finder software can be used for image analysis. We do provide a GAL file (GenePix Array List) for each array, which can be used to set up grids for image analysis. You can find more information about GAL files at http://www.moleculardevices.com/pages/software/gn_genepix_file_formats.html#gal.