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Antibody Array Overview and Protocol

Protocols & Procedures

Tips and Tricks

Reconstitution & Storage Instructions  
Western Blotting/Immunoblotting (WB/IB) Protocol WB/IB Tips & Tricks
Immunohistochemistry (IHC) Protocol IHC Tips & Tricks
Immunocytochemistry (ICC) Protocol ICC Tips & Tricks
Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol ELISA Tips & Tricks
Blocking Peptide Competition Protocol (BPCP)  
Immunoprecipitation (IP) Protocol IP Tips & Tricks
Antibody Array (AA) Protocol

About Aviva:

Aviva is an antibody production company with offices in the Sorrento Valley, San Diego biotech community. We have manufactured over 30,000 antibodies to date. Many are currently on the website at www.avivasysbio.com, but we have others available which we may consider for future testing. Please note that 100% of our antibodies on www.avivasysbio.com are manufactured and quality control tested by us and are in inventory in our San Diego facility. Aviva’s extensive catalog of high-quality antibodies positions the company well to fuel antibody array development.

For more information, please email us atinfo@avivasysbio.com

Overview of Aviva Systems Biology’s antibody collection, production platform and potential areas for collaboration:

● >15,000 antibodies in inventory at our San Diego facility. We believe this makes us one of the largest polyclonal antibody producers in the market.
● 200 - 300 New Antibodies Released Per Month

Antibody Collection is Focused on:

● Unique Content Not Available Commercially From Other Vendors
● Largest Collection of Transcription Factor Antibodies (>4,000)

Unique Bioinformatic tools available to Researchers:

● Antibody Catalog Linked to Species Reactivity and Disease Type
● Entire Antibody Catalog Searchable by User-Defined Protein Sequence

Enhanced Antibody Validation:

● 100% of our antibodies are quality control tested by Western Blot
● >1,000 Antibodies Currently Available with IHC Data
● Monoclonal Antibody Production is underway
● Custom pAb and mAb production and validation services available

Interested in obtaining partners for:

● Collaborative Assay, Kit and New Platform development
● Antibody sourcing for assays, kits and screening platforms (microarrays, ELISA, Luminex, SPR, etc.)


For more information, please email us atinfo@avivasysbio.com

Antibody Arrays

Description:

An antibody array allows for the screening/profiling of multiple proteins within a samples entireproteome. The methodology behind antibody arrays is much like that of an ELISA.Antibodiesare immobilized and bound to the slide support through covalent interactions.Proteinsare extracted from control and experimental samples and labeled with appropriate conjugate (biotin or biotin-fluorophore). After cleanup to remove any unbound, free label, the proteins are allowed to bind to antibodies on the array (membrane or glass). Once bound, the array can be visualized for those proteins labeled with a biotin-fluorophore. If proteins were labeled with biotin alone, visualization is done through fluorescence or chemiluminescence, usually involving a streptavidin conjugate. Depending on whether a membrane or glass slide array is used, visualization can be done using a microarray scanner or by exposure to X-ray film. Each array includes positive, negative and internal controls. Redundancy in the number of antibodies spotted onto the array ensures effective and accurate results.

Equipment:

● Vortex
● Centrifuge
● Orbital Shaker
● Microarray Scanner (DNA scanner may substitute)

Reagents Needed:

● 1X PBS (pH 7.4)
● Extraction buffer
● Lysis buffer
● Lysate purification column
● Biotin reagent
● Dimethylformamide (DMF)
● Labeling buffer
● Biotin labeling stop reagent
● Blocking solution
● milli-Q or distilled water
● Antibody Array Slides (coated with nitrocellulose and spotted with antibodies)
● Coupling reagent (antibody probe 0.3-0.6 mg/ml)
● Streptavidin reagent
● Detection buffer

Sample Preparation: Proteins from a variety of sources can be used in antibody arrays.

Cells

1. Obtain 1-5 million cells from culture and wash with cold (4oC) 1 X PBS 3 times
2. Centrifuge cells at 4oC and remove supernatant
3. Re-suspend pellet in extraction buffer or lysis buffer. For 1-2.5 million cells add 100 uL of extraction buffer and for 2.5-5 million cells add 200 uL of extraction buffer
4. Vortex briefly, but make sure all of pellet has been re-submerged and mixed into solution
5. Incubate cells (in extraction or lysis buffer) on ice for about an hour. Mix occasionally during incubation
6. Centrifuge for about 15-20 minutes at 4oC and collect supernatant

Tissue

1. Obtain 10-40 mg of tissue and add extraction buffer (100 uL extraction buffer for 10-20 mg of tissue, 200 uL for 20-40 mg tissue)
2. Keep sample on ice and homogenize thoroughly
3. Centrifuge for 15-20 minutes at 4oC

Lysate Purification and Quantification– Column purification of lysate further purifies protein sample by removing unwanted buffer and reagents

1. Column purification using proprietary column
2. Measure UV absorbance of lysate and verify that it is greater than 4.0 OD (280nm); ideally, you want protein concentration to be 2-10 ug/uL


Protein Labeling

1. Add Biotin reagent to 100 uL of DMF
2. Add 25-40 uL of labeling buffer to protein sample (10-25 uL of sample or 40-100 ug of protein). Labeling buffer and protein sample should equal around 50 uL
3. Add 1.5 uL of Biotin/DMF to protein sample
4. Agitate gently for 2 hours at room temperature
5. After 2 hours, add 25 uL of stop reagent and incubate for another 30 minutes


Blocking

1. Warm slides and blocking solution mix (with appropriate amount of dry milk added) to room temperature
2. Submerge glass slide in blocking solution for 45 minutes with gentle agitation
3. Wash slide using milli-Q or distilled water 5-10 times, thoroughly
4. Make sure to shake off excess water before continuing to next step


Coupling

1. Mix appropriate amount of coupling reagent (with dry milk added) to 40-100 ug of Biotin labeled proteins
2. Of this mixture, pour solution into a well and completely submerge slide in solution
3. Incubate with gentle agitation for an hour. Note: if working with nitrocellulose membrane instead of glass slide, may need to incubate overnight
4. Wash slide with 1X wash solution for 10 minutes with gentle agitation. Repeat twice more
5. Rinse glass slide with milli-Q or distilled water 5-10 times, thoroughly. Shake excess water off


Detection

1. Add Streptavidin reagent to appropriate amount of detection buffer
2. Allow slide to incubate in Streptavidin solution for 45 minutes with gentle agitation. Note: shield from light; make sure petri dish or slide container is covered with aluminum foil or incubation is carried out in a dark room
3. Rinse slide with 1X wash solution twice (for ten minutes each with gentle agitation)
4. Slide can be dried through centrifugation or by using compressed air/nitrogen
5. Use microarray scanner or similar device for detection. Note: if using a nitrocellulose membrane with streptavidin-HRP, expose membrane to x-ray film for 1-10 minutes

Antibody Array FAQ:

What researcher needs are addressed by antibody arrays?

● Compare the expression levels of many proteins in one cell/piece of tissue at a time
Protein ExpressionProfiling (quantitative + qualitative)
● Measure changes in phosphorylation at specific sites
● Look at differences between normal, diseased, or treated samples
● Identification of biomarkers
● Development of drug compounds

What kind of samples can I test on arrays?

● Prepared cells, tissue or serum

Are array results quantitative or qualitative?

● Antibody arrays provide mainly qualitative information aboutprotein expression. This ratio is produced by comparing one sample’sprotein expressionto a reference sample

How is background dealt with?

● Deal with blocking issues by making sure slides are rinsed extensively with distilled water after blocking reagent



What is the range of the # of antibodies/array?

● Dependent on array type, range is as little as 10 antibodies/ array to over 200 antibodies/array

Membrane Arrays vs. Glass Arrays

Membrane Array

Glass Array

Less proteins can be detected per membrane (less antibodies on membrane)

A larger number of proteins can be screened for in one glass chip (versus a membrane)

More Expensive

Less Expensive

Longer assay time

Shorter time for assay

Mainly Chemiluminescent Detection

Mainly Fluorescent Detection

More sample needed for detection

Less sample needed for detection

What data is generated and how is it analyzed?

● Results of detection are typically based on signal intensity, average intensity (for replicate spots), coefficient of variation for replicate spots, ratio of protein signal in a diseased sample versus a control sample

Examples of antibody array utility:

Disease detection- Disease/condition-specific antibody arrays exist for kidney injury, fertility hormones array, cardiac arrays, drugs of abuse arrays, growth promoter arrays, synthetic steroid arrays, etc.

http://www.ncbi.nlm.nih.gov/pubmed/19177163

This effort attempts to identify biochemical markers for screening and early intervention in women for psychosocial stress using biochip arrays. Plasma concentrations of 17 different cell signaling proteins were measured in 195 women on long-term sick-leave for a stress-related affective disorder, 45 women at risk for professional burnout and 84 healthy women. Significant increases in the levels of MCP-1, VEGF and EGF were found to be potential markers for screening and early intervention of women for prolonged psychosocial stress.

Biomarker analysis– Identify key components of certain diseases at different stages of development

http://www.jci.org/articles/view/40582

Metastasis is a biological cascade of multiple steps: loss of cellular adhesion, increased motility and invasiveness, entry and survival in the circulation, exit into new tissue, and eventual colonization at a distant site. This suggests that cells containing metastatic lesions would have to accumulate expression of multiple, if not all, genes necessary for successful execution of the metastatic cascade from the primary tumor. Therefore, important and long-standing questions that remain concern the identity of genes that mediate these metastasis-promoting processes. Identification and characterization of these genes will not only shed new insight into the molecular basis of cancer metastasis but also inform therapeutic strategies to improve the outcome of treatment of human cancers. Arrays were used in this paper to give insight into factors that promote metastasis of human cancer cells.

Pathways analysis– Various players in different signaling pathways can be monitored (e.g., cytokine profiling, insulin signaling, angiogenesis, phosphorylation, etc.)

http://onlinelibrary.wiley.com/doi/10.1002/prca.200900020/abstract

The development of insulin resistance and type 2 diabetes is determined by various factors, including defects within the insulin signaling pathway. These mechanisms are still largely unresolved because of the complexity of the molecular events. In this study, an expression and activation state profiling of multiple known key signaling biomolecules involved in insulin metabolic and mitogenic signaling pathways was evaluated using a phosphospecific antibody array platform. This insulin signaling antibody array provides a powerful and effective way to investigate the mechanism of insulin resistance and likely assist the development of innovative therapeutic drugs for type 2 diabetes.

Experimental– Choose species specific array available from array suppliers, discover interaction partners, etc.

http://www.ncbi.nlm.nih.gov/pubmed/17407973?dopt=Abstract

Ginseng has been shown to inhibit cancer cell proliferation and tumor growth, however the mechanisms underlying this inhibition have yet to be elucidated. Using antibodymicroarrays, it was determined that several key cell survival proteins were altered in Ginseng-treated cells, including several members of the mitogen-activated protein kinase (MAPK) family. These results suggest that American ginseng may act to inhibit breast cancer cell proliferation by increasing the expression of RKIP, resulting in inhibition of the MAPK pathway. This novel mechanism has implications in the potential prevention and treatment of breast cancer.

References

http://en.wikipedia.org/wiki/Antibody_microarray

http://www.raybiotech.com/comparison.asp

http://www.fullmoonbiosystems.com/protocols/AntibodyArrayUserGuide.pdf

http://www.fullmoonbiosystems.com/Products/AntibodyArray.htm

http://www.sigmaaldrich.com/life-science/cell-biology/protein-arrays/antibody-arrays/panorama-ab-microarray.html

http://www.cellsignal.com/tools/pathscan_arrays.html

http://www.biomax.us/antibody-arrays.php

http://www.raybiotech.com/G_Series.asp

http://www.rndsystems.com/product_detail_objectname_ProteomeProfilerArray.aspx

For more information, please email us atinfo@avivasysbio.com