Genepix 4200a microarray scanner

Biotin–emodin was synthesized as described before [30 (link)]. Proteome microarrays were first treated with blocking buffer (1% BSA in 0.1% Tween 20 TBST) for 1 h at room temperature. Biotin–emodin and biotin were added to 10 μM in blocking buffer and incubated on the proteome microarray for another 1 h. After that, microarray was incubated with Cy3-Streptavidin dilution (1 : 1000, Sigma-Aldrich CN, Shanghai, China) for 1 h and was spun dry at 250 × g for 3 min. A GenePix 4200A microarray scanner (Molecular Devices, San Jose, CA, USA) was used to visualize and record the results. An R script was applied to process the protein microarray data. The signal-to-noise ratio (SNR) was defined as the ratio of the median of foreground signal to the median of background signal. Another index, “Ratio,” was defined as SNR (biotin-emodin)/SNR (Biotin) [24 (link)].

The preparation of human protein microarrays and the synthesis of biotin-bufalin were carried out as previously described [
28 (link),
29 (link)]. The samples were sealed in buffer [1% bovine serum albumin (BSA) and 0.1% Tween 20 (TBST)] and stirred gently at 25°C for 1 h. Biotin-bufalin was diluted to 10 μM in blocking buffer and incubated at 25°C for 1 h on a proteome microarray. The biotin-bufalin was washed with thiobarbituric acid for 3 times (5 min each), and then incubated with Cy3-streptavidin (Smart-Lifesciences, Changzhou, China) at 1:1000 for 1 h at 25°C, followed by washing with thiobarbituric acid for 3 times (5 min each). The microarrays were dried at 250
g for 3 min and scanned with a GenePix 4200A microarray scanner (Molecular Devices, San Jose, USA), after which the results were recorded. GenePix Pro-6.0 software was used for data analysis.

Proteome microarrays were blocked with blocking buffer (5% BSA in 0.1% Tween 20 TBST) for 1.5 h at room temperature with gentle agitation. SP and CP samples were diluted to 1 μg/ml and incubated on the proteome microarray at room temperature for 2 h. The microarrays were each washed with TBST three times for 5 min, and followed by three quick washes in ddH₂O. The microarrays were spun dry at 250 g for 3 min and were scanned with a GenePix 4200A microarray scanner (Molecular Devices, CA, USA) to visualize and record the results. The proteome microarray data were extracted with GenePix Pro 6.0 (Molecular Devices, CA, USA) and processed as previously described (Wang et al., 2012 (link)).

A collection of 308 GST recombinant fusion proteins were expressed and purified using Glutathione Sepharose beads (GE Healthcare) as described [56 (link), 57 (link)]. The GST-tagged methyllysine reader domains were arrayed onto nitrocellulose-coated glass slides (Oncyte Avid slides, Grace Bio-Labs) using a pin arrayer (Aushon). GST proteins were printed in duplicate as indicated in Additional file 1: Fig. S1. C-terminal biotinylated LIG1_(118–130)K126me0, LIG1_(118–130)K126me0, or H3_(1–20)K9me2 peptides were fluorescently labeled with Cy3-streptavidin and then 100 µg of each peptide was hybridized on the microarrays in 1.8 mL of PBST with 3% milk and 3% bovine serum albumin. Binding of fluorescent peptides was visualized using a GenePix 4200A Microarray Scanner (Molecular Devices), scanned at 10 µm resolution, and quantified using ArrayNinja [26 (link)]. Data shown are normalized within each peptide dataset to the brightest signal. Histone peptide microarray experiments were performed as described [4 (link)], with the following exceptions: proteins were hybridized at 1 µM for 1 h and all hybridization steps were performed at room temperature. Raw data for both reader and histone peptide microarrays are available in Additional file 2: Table S1 and Additional file 3: Table S2.