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Protein Arrays - Application
Differential protein expression profiling
One of the many applications of protein arrays is to complement differential protein expression profiling by other methods, such as 2-D DIGE.
In this example, proteins analyzed by 2-D DIGE would provide targets for raising specific antibodies, which could be used for profiling with protein arrays.
Demonstration of differential protein expression profiling
Binding specificity
Slides spotted with panels of different antibodies were probed with Cy5-labelled prothrombin. High levels of binding were observed to the anti-prothrombin panel. Binding to non-cognate antibodies was very low.
Detection sensitivity
Detection sensitivity was evaluated using a simple sample (prothrombin antigen in buffer) and a complex sample (prothrombin in sheep serum). Detection limits of 10 pg/ml were observed with simple samples. Detection limits of 1–10 ng/ml were observed with complex samples, comparable to detection of proteins on 2-D gels.
To look at differential labelling, slides were probed with decreasing amounts of Cy3-labelled antigen (prothrombin, 100 ng/ml to 10 pg/ml) in the presence of a constant amount of Cy5-labelled prothrombin (10 ng/ml).
With a simple sample (prothrombin in buffer), observed ratios were very close to expected ratios over four orders of magnitude. For the complex sample (prothrombin in serum), deviations in the observed ratios occurred at antigen concentrations below the detection limit.
Differential analysis
Different concentrations of cytokines were spiked into sheep serum samples (samples A and B). Differences between the spiked samples were compared on arrays by two-color differential analysis.
| Cytokine | Sheep serum sample A* | Sheep serum sample B* |
| IL1a | 12 ng/ml | 48 ng/ml |
| IL6 | 48 ng/ml | 12 ng/ml |
| IL1b | 12 ng/ml | 48 ng/ml |
* Sample concentration was 600 µg/ml.
Sample A was labelled with Cy3 and sample B was labelled with Cy5. The samples were combined and used to probe the array. The separate Cy3 and Cy5 images and the overlaid images are shown below.
The Cy3/Cy5 overlay shows that the combined sample contains higher levels of Cy5 IL1a and IL1b relative to Cy3 IL1a and IL1b and lower levels of Cy5 IL6 relative to Cy3 IL6.
The observed differences were generally in close agreement with the expected values. The differences were reproducible as demonstrated in the reverse experiment (data not shown).
Differential analysis of spiked sheep serum samples on protein arrays. The array comprises 39 antibodies and includes positive and negative controls, as well as Cy3/Cy5 BSA standards. Cy3 fluorescence is represented in green and Cy5 fluorescence in red.
Model system—two-color labelling of ECV304 supernatants
Cytokine release was induced by IL1 stimulation of the ECV304 urinary carcinoma cell line (1).
The levels of cytokines in the cell supernatants were validated by ELISA before detection using protein arrays. This cell line is a well-documented model for a number of disease processes such as angiogenesis, lung-airway immune responses and blood-brain barrier co-culture systems.
The two main cytokines released from the carcinoma cell line were IL-6 and IL-8. Changes in the levels of these cytokines over time could be observed using differential analysis of the supernatants on arrays (IL-6 data shown). This response is confirmed in the ELISA analysis.
Arrays were probed with Cy5-labelled supernatants from different time points and Cy3-labelled supernatant from the 5 h time point. The overlaid images are shown (Cy3 fluorescence is green, Cy 5 fluorescence is red). Cy3/Cy5 pre-labelled BSA markers are included.
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ELISA evaluations of the same ECV304 supernatants
Conclusions
§Labelling with CyDye fluors provides high-sensitivity detection of labelled proteins.
§Sensitivity on protein arrays using direct labelling is currently comparable to detection limits of fluorescently labelled proteins on 2-D gels.
§High specificity is achievable using protein arrays.
§Protein arrays can enable differential analysis of complex protein samples.
Reference
1. Suda, K. et al., In Vitro Cell Dev Biol Anim. 37, 505–14. |  |
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