The buffer used for spotting proteins for protein arrays is a critical factor in obtaining reliable and consistent results. Most important impact is on protein aggregation, which can affect spot morphology, and protein binding to the substrate.
Grace Bio-Labs has developed a Protein Arraying Buffer designed to work with our ONCYTE® porous nitrocellulose film slides for optimal spot morphology and protein binding. Our recent results demonstrate the use of GBL Protein Arraying Buffer for optimal protein binding on ONCYTE films, minimal protein aggregation and improved stability of reconstituted proteins during storage between spotting runs.
Figure 1. Microarray protein binding of spotted IgG reconstituted in GBL Protein Arraying Buffer compared to 3 commercially available protein arraying buffers (A, S, and W). Protein binding was determined by fluorescence of an incorporated TRITC label on the spotted protein using a Molecular Devices GenePix 4000B Microarray Scanner at 532nm and are expressed as percent bound normalized to the GBL buffer. IgG samples were reconstituted in respective buffers from a 2x IgG stock solution to ensure equivalent protein spotting concentrations (250 µg/ml final). All buffers were spotted in parallel on ONCYTE AVID porous nitrocellulose slides using a Scienion S3 array spotter. Slides were blocked with Super G Blocking Buffer and washed successively in 1x PBST and 1x PBS (3 times, 5 min. each respectively). (N = 160 replicates per buffer)
Figure 2. Microarray image of various immobilized cytokine proteins spotted in parallel on ONCYTE AVID porous nitrocellulose slides. Cytokines were reconstituted in 1x PBS or GBL Protein Arraying Buffer from freshly reconstituted 2x stock solutions to ensure equivalent spotting concentrations (1 mg/ml spotting concentration). Slides were assayed with primary antibodies directed against their antigen followed by secondary labeling with Alexa-fluor 555 and 647 antibodies. All data shown were collected using a Molecular Devices GenePix 4000B microarray scanner at 532 nm (33% laser power, 400 PMT) and 635nm (33% laser power, 475 PMT).
Figure 3. Presented are data and representative microarray spots from immobilized IgG (250 μg/ml) on ONCYTE AVID slides. IgG samples were prepared in either PBS with tween or GBL Protein Arraying Buffer followed by storage at either 4oC or -20oC for 30 days. Slides were printed with stored IgG and with freshly prepared IgG in the respective buffers. Following printing, slides were assayed with anti-IgG Alexa-Fluor647. All data shown were collected using a Molecular Devices GenePix 4000B microarray scanner at 635nm (33% laser power, 390 PMT). (N = 384 replicates per buffer)