Supernatants were mixed with an equal volume of 2 mM vanadate in PBS and filtered through a low protein binding glass fiber filter.
Probes consist of a redundant group of 9932 PCR-derived, sequence-verified cDNA features (representing ∼6000 unique genes) spotted onto amine-binding glass slides (details available on The Wellcome Trust Sanger Institute Microarray Facility homepage (http://www.sanger.ac.uk/Projects/Microarrays/)).sanger.ac.uk/Projects/Microarrays/
Cleaning of glassware used in the experiments was done as follows: first, it was washed with detergent solution, rinsed with tap water, rinsed with 10%% nitric acid, rinsed with tap water, and finally rinsed with distilled water to prevent metal binding to glasses.
Quartz glass was functionalized with PEG-biotin and PEG (1∶40 in a mass ratio, Laysan Bio), while cover glass was functionalized only with PEG to minimize the nonspecific binding of the DNA substrates or proteins [28].
The purified plasmid had a 260/280 nm ratio of 1.88 indicating that the capacity and specificity of DNA binding to glass filters was not compromised by a low concentration of plasmid in the presence of large amounts of impurities.
Peptide solutions were made in plastic tubes or cuvettes to minimize loss of peptide at low concentrations due to binding to glass surfaces.
Gram staining offered rapid validation of enhanced binding on the glass surface.
The pedestal surface was coated with casein to prevent α-actinin-coated beads from nonspecific binding to the glass surface.
The GMR driver lines used a pentameric arrangement of an enhancer region of the Rh1 promoter (glass binding site).
Initially, we investigated the DNA binding capacity of glass filters with 0.7 um and 1 um pore size and found that more plasmid was isolated using the 30 mm glass filter with a 0.7 um pore-size (Data not shown).
The remaining nucleotides consist of a guanine to which a thiol group is added and 5 T's for flexibility of the probe following binding to the glass slide.
