Protein Domain Microarrays Core
Our core facility utilizes high quality protein domain microarrays, originally developed by the Bedford Laboratory, as a valuable technology to screen for protein-protein interactions. These high-density microarrays consist of purified fusion proteins immobilized on a nitrocellulose coated glass slides (FAST slides); one interesting characteristic of the protein domains present on the microarrays is that they generally retain their native structure, thus resembling their in vivo conformation.
Our protein domain microarrays are generated using two state-of-the-art microarray printers, Flexis Robot (Genomic Solutions) and OmniGrid Accent Microarrayer (Digilab). We put great effort in quality control of the GST fusion proteins as well as consistency of the microarrays’ functionality, aiming to provide a reliable, high-throughput technology to facilitate the identification of novel protein-protein interactions.
Our currently provided protein domain microarrays are:
- Chromatin Associated Domain Array (CADOR)
- Phospho-Serine/Threonine Array
- Phospho-Tyrosine SH2 Binding Domain Array
The use of modified peptides on microarrays:
We commonly use modified peptides to screen for protein-protein interactions. One good aspect of the microarray system is the little amount of peptide required – our largest array uses only 10 ug of peptide per probe. We request our collaborators provide 10 ul of water-solubilized peptide at a concentration of 5 ug/ul for probing purposes.
These peptides must be biotinylated and about 20 amino acids in length, with the modification in the middle of the peptide sequence. Most of the peptides we work with have been synthesized by the W. M. Keck Biotechnology Resource Laboratory at Yale University.
To generate a peptide probe, we pre-conjugate the biotinylated peptide with fluorescently-labeled streptavidin. This probe is incubated on a protein domain microarray and finally scanned using the GenePix Scanner (Axon Instruments). For more information, please visit our Microarray Protocols. In addition to using peptides as probes, other approaches can be used as seen below.
The use of a protein domain microarray in four different screening approaches:
• Synthetically generated histone tail peptides harboring different degrees of lysine methylation.
• Enzymatically modified nucleosomes which more closely resemble the in vivo setting.
• Peptides containing lysine methylation from non-histone proteins.
• Methylated peptides containing glycine- and arginine-rich patches (GAR motifs) to search for methyl specific protein-protein interactions.
Methylated Histone Tails Bind Chromo, MBT, and Tudor Domains
CADOR chips probed with Cy3 labeled peptides that were mono-, di-, or tri-methylated at lysines 4 and 9 of histone H3 and lysine 20 of histone H4. Chromo domain interactions are blocked with a white square. Tudor domains are highlighted with an oval: PHF20 (red) and JMJD2A (turquoise). MBT domain interactions are marked with rectangles: PHF20L (orange) and L(3)MBTL (yellow).
Kim J, Daniel J, Espejo A, Lake A, Krishna M, Xia L, Zhang Y, Bedford MT. Tudor, MBT and chromo domains gauge the degree of lysine methylation. EMBO Rep 7:397-403, 4/2006.
Enzymatic Modified Nucleosomes
Cy3 labeled biotinylated recombinant nucleosomes were incubated with the CADOR protein array. To generate methylated nucleosomes, H2B was biotin-tagged and assembled in nucleosomes which were then modified in vitro using the Suv4-20h2 and G9a HMTase.
Schotta G, Sengupta R, Kubicek S, Malin S, Kauer M, Callen E, Celeste A, Pagani M, Opravil S, De La Rosa-Velazquez IA, Espejo A, Bedford MT, Nussenzweig A, Busslinger M, Jenuwein T. A chromatin-wide transition to H4K20 monomethylation impairs genome integrity and programmed DNA rearrangements in the mouse. Genes & Dev 22:2048-2061, 2008.
Peptides containing Lysine Methylation from Non-Histone Proteins
Top panel shows a CADOR array probed with an anti-GST primary antibody and detected with a AlexaFluor555-conjugated secondary antibody, the section blocked with a white square is shown below along with the peptides used to probe the array; Cy3 labeled peptides p53K370me0, p53K370me1, p53K370me2 and p53K370me3. The K370me2-dependent interaction with double Tudor domains of 53BP1 is circled.
Huang J, Sengupta R, Espejo AB, Lee MG, Dorsey JA, Richter M, Opravil S, Shiekhattar R, Bedford MT, Jenuwein T, and Berger SL. p53 is regulated by the lysine demethylase LSD1. Nature 449:105-109, 2007.
Methylated Peptides containing Glycine- and Arginine-rich Patches (GAR motif)
The array was probed with Cy3 labeled symmetrically arginine methylated peptides from the splicing factor SmD3 (SmD3-Rme2s), which has previously been demonstrated to bind to the Tudor domain of SMN. Fluorescently labeled GAR glycine/arginine motif peptides harboring either aDMA or sDMA residues are probed on a focus microarray that has a selection of Tudor-domain-containing proteins: Pombe, SMN, TDRD3, SPF30 and 53BP1.
Bedford MT. Arginine methylation at a glance. J Cell Sci 120:4243-4246, 2007.