Preserving Protein Immuno-Reactivity

IN Featured Research, ONCYTE nitrocellulose film slides, Product Applications, Super G, SuperNOVA, Uncategorized

preserving_proteinMicroarrays deposited onto solid supports are robust platforms for performing antibody capture and antigen detection assays.  Among various substrates, thin film porous nitrocellulose cast on glass surfaces has excelled in the field of proteomics since its invention by scientists at Grace Bio-Labs (McGrath, CM, Grudzien, J and A. Levine. Cell Vision 2:499-509, 1995). The irregular 3-dimensional microstructure of porous nitrocellulose creates an environment which promotes retention of a protein’s native conformation, thus making it the predominant immobilization surface for protein microarray applications (Balboni et al. Annu Rev Immunol 24:391-418, 2006; Sakanyan. J Chromatography B815:77-95, 2005; Schweitzer et al. Proteomics 3:2190-2199, 2003; Feilner et al. Current Proteomics 1(4):293-295, 2004). 

Nitrocellulose holds significant advantages as a protein substrate, but due to their heterogeneous nature some proteins require additional stabilization.  Recent work in our laboratory indicates that some proteins spotted and immobilized on nitrocellulose film can lose reactivity within weeks of being printed.  For prolonged storage (6 months or more), as much as 35 – 70% of the immobilized protein may lose immunoreactivity depending how they are preserved and stored.

 Grace Bio-Labs recently developed Super G Blocking Buffer which allows for extremely efficient blocking of our ONCYTE Film Slides. To address the issue of protein stability for labile proteins, we have augmented our Super G formulation with components optimized for long-term protein preservation. Here we present data showing how Super G Plus improves assay performance for various proteins after immobilization on nitrocellulose film slides.  Use of this combination blocking buffer and preservative helps retain sensitive detection of proteins over time and shows its effectiveness for antibody capture arrays as well as antigen arrays.

Figure 1. Preservation with Super G Plus increases assay signal of stored protein arrays.

Preservation with Super G Plus increases assay signal of stored protein arrays

Figure 1. Microarray images for immobilized TNFa and TNFb processed after various storage times.  Arrays were treated with Super G Plus or Super G (no preservation) for 1 hr. prior to storage.  Per panel, spots are from slides processed immediately (left), after 1.5 months (center, 7 days @ 38°C accelerated), and after 12 months (35 days @ 38°C accelerated) under ambient humidity conditions.  All data shown were collected using a Molecular Devices GenePix 4000B microarray scanner at 532nm (10% laser power, 355 PMT).  All images are shown at the same contrast (67) and brightness settings (68).

Figure 2. Effects of Super G Plus treatment on protein antigenicity after extended storage.

 

Effects of Super G Plus treatment on protein antigenicity after extended storage.

 

Figure 2. Assay results for various immobilized proteins stored after treatment with Super G Plus or Super G alone (no preservation).  Proteins were spotted on SuperNOVA nitrocellulose film slides, preserved, and stored until processing.  Antigen preservation was performed with a 1 hr. incubation followed by air drying prior to storage.  Storage was performed under accelerated conditions at 45°C for up to 21 days (equiv. to 1 yr. at 4°C) under desiccated conditions.  Controls were assayed immediately after preservation incubations.  Assays were performed with individual anti-antigen antibodies and detected by fluorescence at 532nm.

 

Figure 3. Effects of Super G Plus and storage conditions on protein immunoreactivity after extended storage.

 

Effects of Super G Plus and storage conditions on protein immunoreactivity after extended storage.

 

Figure 3. Assay results for IFN-g and IL1b stored after treatment with Super G Plus or Super G alone (no preservation) under desiccated or ambient humidity conditions.  Proteins were spotted on SuperNOVA nitrocellulose film slides, preserved, and stored until processing.  Antigen preservation was performed with a 1 hr. incubation followed by air drying prior to storage.  Storage was performed under accelerated conditions at 45°C for up to 21 days (Desiccated) or 38°C for up to 35 days (Non-Desiccated), both equivalent to 1 year storage at 4°C.  Controls were assayed immediately after preservation incubations.  Assays were performed with individual anti-antigen antibodies and detected by fluorescence at 532nm.