Journal: Nature protocols

Article Title: Quantifying protein-protein interactions in high throughput using protein domain microarrays

doi: 10.1038/nprot.2010.36

Figure Lengend Snippet: Quantifying domain–peptide interactions in high throughput using protein domain microarrays. (a) A set of n protein interaction domains are cloned, expressed, purified and arrayed. The microarrays of protein domains are then probed with m fluorescently labeled peptides to reveal the full n × m matrix of domain–peptide interactions. (b) For high-affinity interactions (KD < 2 μM), dissociation constants can be determined directly using protein microarrays. Microarrays of protein domains are probed with eight concentrations of each peptide and the resulting saturation binding curves are used to determine the binding affinity of each domain–peptide interaction. (c) For low-affinity interactions (KD < 50 μM), microarrays of protein domains are probed with fluorescently labeled peptides and a fluorescence threshold is used to divide domain–peptide pairs into putative interactions (array positives) and putative noninteractions (array negatives). A secondary assay (FP) is then used to retest and quantify all array positives. The result is a quantitative interaction data set (data set 1) in which all the false positives in the microarray data set have been eliminated. To remove false negatives, it is necessary to build a model that can predict domain–peptide interactions. The model is then used to highlight suspected false negatives in the microarray data set, which are retested by FP. By performing multiple cycles of prediction, retesting and retraining of the model, many of the microarray false negatives can be corrected. This results in a substantially refined data set (data set 2).

Article Snippet: Custom-made aldehyde-coated 74.5 mm × 112.5 mm × 1 mm glass substrates (Thermo Fisher Scientific Inc., cat. no. HAR-1101-C60) ProPlate Gaskets (Grace Bio-labs, cat. no. 204971) 96-Well No-Bottom microtiter plates (Greiner Bio-One, cat. no. 655000) 96-well microtiter plates (Greiner Bio-One, cat. no. 650201) 384-well assay plates, black nonbinding, for FP assays (Corning, cat. no. 3575) 384-well microarray plates, for printing protein microarrays (Genetix, cat. no. X7022) Storage Mat III (Costar, cat. no. 3080) Disposable reagent reservoirs, sterile (VWR, cat. no. 82026-350) Deep-well 96-well microtiter plate (Costar, cat. no. 3960) 14 ml Polypropylene Round-Bottom Tube (Becton Dickinson, cat. no. 352059) NanoPrint LM60 Microarrayer (Arrayit Corporation) including cooling block for source plate and destination block designed for 16 microtiter-sized glass plates Silicon Microarray spotting pins (Parallel Synthesis Technologies Inc., cat. no. SMT-S75) 48-pin Silicon printhead assembly (Parallel Synthesis Technologies Inc., cat. no. SMT-H192) Funnel for silicon 48-pin printhead (Parallel Synthesis Technologies Inc., cat. no. SMT-F48) Fluorescence microarray scanner—Tecan LS400 Laser Scanner or similar (Tecan, cat. no. LS400) ArrayPro software or equivalent (Tecan) Matlab software or equivalent (The MathWorks) Peptide Synthesizer, Apex 396 or similar (Aapptec, cat. no. Apex 396-DC-FW-M) Kromasil 100 (5 μm) C18 semi-prep column (Peeke Scientific, cat. no. 100-5-C18 20 × 250) Kromasil 100 (5 μm) C18 analytical column (Peeke Scientific, cat. no. 100-5-C18 2.1 × 150) Superdex 200 10/300 GL column (Amersham Biosciences, cat. no 17-5175-01) Fluorescence polarization plate reader, Analyst AD 96:384 or similar (LJL Biosystems) Storage Mat Applicator (Corning, cat. no. 3081) Microtiter plate shaker (Lab Line, cat. no. 4625) Floor centrifuge, capable of holding both 500 and 30 ml tubes 2-L Baffled flasks (VWR , cat. no. 89083-696) 500-ml Centrifuge tubes (Sorvall, cat. no. 7-9957) 30-ml Oakridge centrifuge tubes (Thermo Fisher Scientific Inc., cat. no. 3119-0010) P1000 multichannel pipette (Rainin, cat. no. L1000) P200 multichannel pipette (Rainin, cat. no. L200) P10 multichannel pipette (Rainin, cat. no. L10) Mini Bunsen burner Agilent 1200 series HPLC with fraction collector

Techniques: High Throughput Screening Assay, Clone Assay, Purification, Labeling, Binding Assay, Fluorescence, Microarray

Journal: Nature protocols

Article Title: Quantifying protein-protein interactions in high throughput using protein domain microarrays

doi: 10.1038/nprot.2010.36

Figure Lengend Snippet: Using protein domain microarrays to identify and quantify domain–peptide binding interactions. (a) Fluorescent images of eight identical SH2/PTB domain microarrays in separate wells of a 96-well microtiter plate. The fluorescence arises from a trace amount of Cy5-labeled BSA that was added to each protein before arraying. (b) Fluorescent images of SH2/PTB microarrays, probed with eight concentrations of a 5(6)-TAMRA-labeled phosphopeptide derived from ErbB2 pY1139 (5(6)-TAMRA-PLTCSPQPEpYVNQPDVR). (c) Plots showing fluorescence as a function of peptide concentration for 28 high-affinity interactions. The data were fit to equation (1) to determine the KD. (d) PDZ domain microarrays probed with fluorescently labeled peptides. Each PDZ domain was spotted in quadruplicate in wells of 96-well microtiter plates (requiring four wells to include all domains). The Cy5 (red) images are used to identify PDZ domain spots. The green images depict arrays probed with fluorescently labeled peptides. (left, 5(6)-TAMRA-NNGSNAKAVETDV, a promiscuous peptide representing the C terminus of Kv1.4 and right, 5(6)-TAMRANNGQSPANIYYKV, a more selective peptide from Ephrin B1/2).(e) FP titration curves obtained for the array positives identified in (d). Panels a–c31 and d–e36 of this figure are reproduced with permission from the publishers.

Article Snippet: Custom-made aldehyde-coated 74.5 mm × 112.5 mm × 1 mm glass substrates (Thermo Fisher Scientific Inc., cat. no. HAR-1101-C60) ProPlate Gaskets (Grace Bio-labs, cat. no. 204971) 96-Well No-Bottom microtiter plates (Greiner Bio-One, cat. no. 655000) 96-well microtiter plates (Greiner Bio-One, cat. no. 650201) 384-well assay plates, black nonbinding, for FP assays (Corning, cat. no. 3575) 384-well microarray plates, for printing protein microarrays (Genetix, cat. no. X7022) Storage Mat III (Costar, cat. no. 3080) Disposable reagent reservoirs, sterile (VWR, cat. no. 82026-350) Deep-well 96-well microtiter plate (Costar, cat. no. 3960) 14 ml Polypropylene Round-Bottom Tube (Becton Dickinson, cat. no. 352059) NanoPrint LM60 Microarrayer (Arrayit Corporation) including cooling block for source plate and destination block designed for 16 microtiter-sized glass plates Silicon Microarray spotting pins (Parallel Synthesis Technologies Inc., cat. no. SMT-S75) 48-pin Silicon printhead assembly (Parallel Synthesis Technologies Inc., cat. no. SMT-H192) Funnel for silicon 48-pin printhead (Parallel Synthesis Technologies Inc., cat. no. SMT-F48) Fluorescence microarray scanner—Tecan LS400 Laser Scanner or similar (Tecan, cat. no. LS400) ArrayPro software or equivalent (Tecan) Matlab software or equivalent (The MathWorks) Peptide Synthesizer, Apex 396 or similar (Aapptec, cat. no. Apex 396-DC-FW-M) Kromasil 100 (5 μm) C18 semi-prep column (Peeke Scientific, cat. no. 100-5-C18 20 × 250) Kromasil 100 (5 μm) C18 analytical column (Peeke Scientific, cat. no. 100-5-C18 2.1 × 150) Superdex 200 10/300 GL column (Amersham Biosciences, cat. no 17-5175-01) Fluorescence polarization plate reader, Analyst AD 96:384 or similar (LJL Biosystems) Storage Mat Applicator (Corning, cat. no. 3081) Microtiter plate shaker (Lab Line, cat. no. 4625) Floor centrifuge, capable of holding both 500 and 30 ml tubes 2-L Baffled flasks (VWR , cat. no. 89083-696) 500-ml Centrifuge tubes (Sorvall, cat. no. 7-9957) 30-ml Oakridge centrifuge tubes (Thermo Fisher Scientific Inc., cat. no. 3119-0010) P1000 multichannel pipette (Rainin, cat. no. L1000) P200 multichannel pipette (Rainin, cat. no. L200) P10 multichannel pipette (Rainin, cat. no. L10) Mini Bunsen burner Agilent 1200 series HPLC with fraction collector

Techniques: Binding Assay, Fluorescence, Labeling, Phospho-proteomics, Derivative Assay, Concentration Assay, Titration

Journal: Nature protocols

Article Title: Quantifying protein-protein interactions in high throughput using protein domain microarrays

doi: 10.1038/nprot.2010.36

Figure Lengend Snippet: Microarrays in microtiter plates. (a) NanoPrint Microarrayer spotting recombinant domains onto aldehyde-displaying glass substrates. (b) Attachment of microarrays to the bottom of a bottomless 96-well microtiter plate using an intervening silicone gasket. Panel b of this figure is reproduced, with permission, from the publisher37.

Article Snippet: Custom-made aldehyde-coated 74.5 mm × 112.5 mm × 1 mm glass substrates (Thermo Fisher Scientific Inc., cat. no. HAR-1101-C60) ProPlate Gaskets (Grace Bio-labs, cat. no. 204971) 96-Well No-Bottom microtiter plates (Greiner Bio-One, cat. no. 655000) 96-well microtiter plates (Greiner Bio-One, cat. no. 650201) 384-well assay plates, black nonbinding, for FP assays (Corning, cat. no. 3575) 384-well microarray plates, for printing protein microarrays (Genetix, cat. no. X7022) Storage Mat III (Costar, cat. no. 3080) Disposable reagent reservoirs, sterile (VWR, cat. no. 82026-350) Deep-well 96-well microtiter plate (Costar, cat. no. 3960) 14 ml Polypropylene Round-Bottom Tube (Becton Dickinson, cat. no. 352059) NanoPrint LM60 Microarrayer (Arrayit Corporation) including cooling block for source plate and destination block designed for 16 microtiter-sized glass plates Silicon Microarray spotting pins (Parallel Synthesis Technologies Inc., cat. no. SMT-S75) 48-pin Silicon printhead assembly (Parallel Synthesis Technologies Inc., cat. no. SMT-H192) Funnel for silicon 48-pin printhead (Parallel Synthesis Technologies Inc., cat. no. SMT-F48) Fluorescence microarray scanner—Tecan LS400 Laser Scanner or similar (Tecan, cat. no. LS400) ArrayPro software or equivalent (Tecan) Matlab software or equivalent (The MathWorks) Peptide Synthesizer, Apex 396 or similar (Aapptec, cat. no. Apex 396-DC-FW-M) Kromasil 100 (5 μm) C18 semi-prep column (Peeke Scientific, cat. no. 100-5-C18 20 × 250) Kromasil 100 (5 μm) C18 analytical column (Peeke Scientific, cat. no. 100-5-C18 2.1 × 150) Superdex 200 10/300 GL column (Amersham Biosciences, cat. no 17-5175-01) Fluorescence polarization plate reader, Analyst AD 96:384 or similar (LJL Biosystems) Storage Mat Applicator (Corning, cat. no. 3081) Microtiter plate shaker (Lab Line, cat. no. 4625) Floor centrifuge, capable of holding both 500 and 30 ml tubes 2-L Baffled flasks (VWR , cat. no. 89083-696) 500-ml Centrifuge tubes (Sorvall, cat. no. 7-9957) 30-ml Oakridge centrifuge tubes (Thermo Fisher Scientific Inc., cat. no. 3119-0010) P1000 multichannel pipette (Rainin, cat. no. L1000) P200 multichannel pipette (Rainin, cat. no. L200) P10 multichannel pipette (Rainin, cat. no. L10) Mini Bunsen burner Agilent 1200 series HPLC with fraction collector

Techniques: Recombinant