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FACS analysis revealed that saturation of binding was achieved by application of 1 2 µg protein to 500,000 cells (Fig. 4A).

Subdomain D1 deletion had little impact on PEPD ErbB2 binding; the binding affinity after removing subdomains D2 and D4 was reduced 3.5- and 51.0-fold, respectively, but full PEPD binding was achieved by raising the PEPD concentration; removing subdomain D3 completely abolished PEPD binding.

PaTu-S and PaTu-T cells grown on glass coverslips for 24 48 h were washed 3 times with PBS, fixed for 15 min in 4% paraformaldehyde and permeabilised in 0.1% Triton- X-100 for 5 min. Blocking of unspecific binding was achieved by a 1 h incubation in 10% Aurion BSA-c (Aurion, Waageningen, The Netherlands).

Binding was achieved by incubating 100 μg of cell-free extract protein with 5 ng of labelled fragment for 15 min on ice.

Binding was achieved by incubating increasing amounts of heterologously expressed, thrombin-cleaved CREI-96 (500 nM, 1000 nM, and 2000 nM) with 4 ng of the labelled, double-stranded DNA fragment in GST elution buffer (10 minutes at 22°C).

Binding was achieved by incubating 100 ng of the GST fusion proteins or 60 ng of in vitro translated, unlabelled 2488prp (putative repressor protein) with 15 ng of labelled fragment for 15 min on ice.

Sequence-specific binding is achieved by in situ hybridization of the primary probes to the genomic DNA.

It remains to be determined exactly how specificity of binding is achieved by ZNF274 but possibilities include heterodimerization with another protein or selective use of a subset of the zinc fingers.

A third method comes close to liposome chromatography: beads are mixed with liposomes and binding is achieved by several cycles of freezing and thawing [ 20].

In T. thermophilus HB27, DNA binding is achieved by pilQ, transported through the outer cell membrane by comEA, pilF and pilA4, through the thick cell wall layers and inner membrane by pilM, pilN, pilO, pilA13 and comEC.

In those that possess a single domain, high affinity DNA binding is achieved by a zinc finger complemented by short stretches of highly basic amino acids flanking the domain [ 45, 46].