ADVANTAGES OF NITROCELLULOSE FILM-SLIDES AND FILM-PLATES FOR PROTEIN/GLYCOPROTEIN MICROARRAYS
Proteins that bind specific DNA sequences play a significant role in gene expression and regulation. Furthermore, the DNA sequences that bind regulatory sequences are highly conserved among species, though not all of the proteins that bind these sites have been identified. Protein arrays provide an excellent screening tool for identifying proteins that bind highly conserved genomic sequences (Hall et al, 2004; Ho et al., 2005). In these studies, oligonucleotides are used to probe a protein array of potential transcription factors. The protein-DNA interactions were detected through a fluorescent tag that was covalently attached to the oligomers prior to incubating with the protein array. A recent study by Hu et al. (2013) exploited the high-throughput power of protein microarrays to examine a large repertoire of human transcription factors and their dependence on methylation for binding DNA. This large study used 154 methylated DNA sequences to probe individual arrays of over 1500 transcription factors and associated proteins. The full-length proteins were expressed with a GST tag and purified by affinity chromatography and then printed on a microarray.
Porous nitrocellulose film is the substrate of choice for protein arrays designed to detect binding partners, largely because of its high protein binding capacity, which can exceed non-porous 2-dimensional substrates such as functionalized glass by 500X and thus provides highly sensitive detection of low abundance proteins. Furthermore, porous nitrocellulose film retains the biological activity (i.e. structure) of the proteins due to its non-covalent capture of the proteins within the porous matrix. Multiple protocols based on nitrocellulose film slides exist for protein arrays and may vary from lab to lab. At Grace Bio-Labs we have developed reagents and protocols designed to obtain consistent and sensitive results with our nitrocellulose film.
Use of Grace Bio-Labs products in Protein-DNA arrays:
As mentioned above, porous nitrocellulose is the substrate of choice for protein arrays. ONCYTE® PNC (Porous NitroCellulose) Microarray Slides and Reagents from Grace Bio-Labs are designed to provide a complete system for sensitive and consistent results for your protein arrays. Our film slides are manufactured with a stringent quality control protocol that ensures minimal variability and consistently low fluorescence background compared to other commercially available film slides.
Incubation chambers from Grace Bio-Labs, including the HybriSlip and ProPlate, provide excellent results with all protein array applications. The incubation chamber is an important parameter that is often overlooked when performing microarray experiments. The ideal chamber should allow for sufficient sample mixing during the assay incubation and wash steps and should minimize the volume of sample required. Active mixing has been shown to significantly affect assay signal and uniformity.
Coverslips generally allow for the lowest sample incubation volumes and, but do not allow sample mixing during incubation. If the use of a coverslip is necessary for your particular assay, we recommend the use of Grace Bio-Labs HybriSlip™ over conventional glass coverslips. In addition, Grace Bio-Labs has developed incubation chambers that facilitate incubations for a wide range of sample volumes with various The Pro-Plate® chamber from Grace Bio-labs is excellent for most microarray applications on slide or plate format.
Ho et al., Linking DNA-binding proteins to their recognition sequences by using protein microarrays. PNAS, 2005: 103(26).
Hall, et al., Regulation of gene expression by a metabolic enzyme, 2004. Science 306, 482–484.
Hu. et al., DNA methylation presents distinct binding sites for human transcription factors, 2013. eLife; 2:e00726.