Reverse phase protein arrays (RPPA) are designed for sensitive quantitation of a target protein in a very small sample of tissue lysate, and multiplex analysis of many samples in one experiment. This technology holds great promise for the identification and analysis of critical biomarkers for disease states in patient samples and for high-throughput drug screening using cultured cell systems. Quantitative assessment of these biomarkers in each cell lysate spot is highly dependent on a robust normalization method across test subjects in a study. A common approach for performing normalization within and across RPPA arrays is to base the normalization factor on the total cell lysate protein present in each spot. This can be accomplished by quantitating the arrayed proteins using stains such as colloidal gold or with SYPRO® Ruby and fluorescence detection (Note: this is typically performed using representative arrays separate than those for antibody biomarker detection). A cost effective, yet sensitive, fluorescent stain method using Fast Green FCF has also been reported for RPPA cell lysate quantitation down to approximately 100 pg protein per spot using near-infrared detection (Loebke, et al., Proteomics 7, 558-584, 2007) where intrinsic fluorescence of nitrocellulose is negligible.
Grace Bio-Labs is developing a near-infrared microarray imager (ArrayCAM, 405nm/800nm) designed for optimal detection on porous nitrocellulose film slides, as discussed in our previous blog article. Using the accompanying method and ArrayCAM, we show sensitive Fast Green protein quantitation down to approximately 8 pg protein per spot on ONCYTE®AVID Porous Nitrocellulose film slides (Fig. 1A and 1B, see attachment for method). As with many protein stains, it should be noted that the degree of staining is dependent on the chemical makeup of individual proteins, as observed in the lower IgG staining compared to Lysozyme and BSA. The results of this are inconsequential for RPPAs where staining of a wide population of proteins from whole cell/tissue lysates would not likely be influenced by differences in individual protein staining.
With ArrayCAM, we also obtained sensitivity down to 8 pg using printed cell lysate arrays (Fig. 2). RPPA cell lysates are typically arrayed as 2-fold dilutions with the most concentrated sample spots containing approximately 1000 – 2000 pg of total protein (1-2 mg/ml cell lysate protein, printed with 1 nl volume). The 8 pg sensitivity obtained with Fast Green/ArrayCAM is more than sufficient for typical RPPA sample quantitation, and is comparable to the sensitivity obtained using SYPRO Ruby with 532nm/575nm detection on a GenePix 4400 (Molecular Devices) which resulted in higher signal intensities but also significantly higher background. The sensitivity reported here for both methods was for the lowest observable cell lysate dilution tested.
This is one of various methods being developed for sensitive biomarker detection on porous nitrocellulose film slides using ArrayCAM. In addition to external normalization methods with Fast Green, use of multichannel fluorescence imagers such as ArrayCAM allow for assay normalization using internal reference standards (ex: β-actin) as well as multi-marker detection on the same microarray spot. For more information regarding porous nitrocellulose and the advantages of near-infrared detection, view our recent presentation at the Microarray World Congress 2013 highlighting these technologies.
Figure 1. Fast Green Protein Quantification on ONCYTE® Film Slides with near IR detection using ArrayCam.
Figure 2. Quantitation of Cell Lysates on RPPA using Fast Green with ArrayCAM produces results comparable to Sypro Ruby Protein Stain measured on a GenePix scanner (Molecular Devices, Sunnyvale, CA).
Figure 1. Total protein may be quantified after spotting on ONCYTE® AVID Film Slides by staining with Fast Green and measuring fluorescence using an ArrayCAM, an imaging system developed at Grace Bio-Labs. ArrayCAM is a CCD-based imaging system using laser excitation at 405 nm. Fast Green emission is 800 nm. (NOTE: Similar results were obtained with ONCYTE® SuperNOVA Film Slides from Grace Bio-Labs.)
A. Shown is a range of standard proteins from 8 – 500 pg after on-slide staining with Fast Green.
B. Detection and quantitation of BSA, Lysozyme, and IgG using a Fast Green staining protocol (see attachment) with ArrayCAM are linear down to the lowest protein deposition (8 pg).
Figure 2. Fluorescent signal for total protein detection with Fast Green/ArrayCAM yields linear results at cell lysate concentrations commonly used for RPPA analysis down to approximately 8 pg protein per spot. Similar results were obtained using a standard protein stain method (SYPRO® Ruby total protein stain; 532nm/575nm detection) with the use of a focused-laser microarray scanner (Molecular Devices GenePix 4400). Cell lysates were from Calyculin A-treated Jurkat cells spotted in 2-fold serial dilution starting with a concentration of 1 mg/ml in a Tris/SDS/Glycerol lysis buffer.
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