Components of Reverse-Phase Protein Array (RPPA) methods and their impact on the overall performance of this assay were presented at the 2nd annual RPPA Conference in Edinburgh, Scotland on November 12. Entire presentation can be viewed here. View poster here.
View here for information regarding our RPPA Assay System
Selecting Surfaces, Reagents, and Detection Methods for Optimal Reverse-Phase Protein Array Assays
RPPA assays have evolved in different laboratories with various protocols that include different binding substrates and blocking buffers, lysis and printing reagents, as well as assay and detection strategies. Current components of the RPPA assay are mostly centered around the use of porous nitrocellulose films and have been optimized to yield robust results. As new reagents and binding substrates continually appear on the market, it is important to assess their usefulness at improving sensitivity with these established methods.
Data presented by Grace Bio-Labs at the Second Annual RPPA Conference in Edinburgh on November 12, 2012, compare the characteristics of various protein binding substrates as they pertain to RPPA. These comparisons indicate that porous nitrocellulose and activated, non-porous nitrocellulose film slides are most effective with standard RPPA methods for obtaining the most compact spot morphologies, highest signals, and best signal-to-noise. Data highlighting the impact of various blocking reagents was also presented, pointing to the importance of using a blocker that is effective and inert to the assay system. Results suggest that RPPA would greatly benefit from use of components designed to work together to achieve optimal results.
RPPA Optimization and Standardization
Grace Bio-Labs invented the nitrocellulose film slide and has optimized this surface for protein binding capacity, porosity, and lower fluorescent backgrounds – making them optimal surfaces for Reverse-Phase Protein Arrays. Additionally, we have developed reagents formulated specifically for our ONCYTE® porous nitrocellulose films to maximize signal-to-noise. Use of Super G Blocking Buffer overcomes a limitation observed with some other blocking reagents which impairs the ability to quantify results with some antibodies. This non-protein blocking reagent promotes detection of various protein markers previously obscured with protein-based blockers due to high antibody-specific backgrounds.
As RPPA progresses into the clinical setting, use of standardized substrates and reagents will facilitate higher quality data for individual assays and produce consistent results between assays, laboratories, and institutions. Here Grace Bio-Labs introduces Reverse-Phase Protein Microarray Kits centered on our ONCYTE® film slides and optimized reagents for RPPA.