A Peptide Microarray Technology for Specificity Profiling of Antibodies
Published: April 30, 2024
Abstract
Source: Cornett, E. M., et al. Analysis of Histone Antibody Specificity with Peptide Microarrays. J. Vis. Exp. (2017).
This video demonstrates a technique that applies peptide microarray technology to profile the specificity of antibodies recognizing post-translational modifications (PTMs) on histone peptides. A microarray slide containing immobilized fluorophore-conjugated histone peptides with known PTM combinations is treated with target PTM-specific antibodies. This process helps identify the antibody's specificity for the target and its potential cross-reactivity with non-target PTMs.
Protocol
1. Designing the Array Slide and Source Plate Layout Under the 'Plan a slide layout' heading on the ArrayNinja interface, click on the link that corresponds to the microarray printer being used. NOTE: ArrayNinja has been programmed to mimic the robotic movement of two commonly used microarray printers (see Table 1). Compatibility with the other arrayers can be configured upon request. Click inside the 'Load plate' dialog …
Representative Results
Figure 1: ArrayNinja Design Module. A screen shot of the ArrayNinja design module is shown in the dotted line. The control panel (top) shows all of the parameters that can be altered on the microarray printer. As these parameters are adjusted, the cartoon image of the slide layout (bottom left) updates in real time. After the layout is set, the user can mouse over individual spots to enter unique…
Açıklamalar
The authors have nothing to disclose.
Materials
| Printing Buffer | ArrayIt | PPB | |
| BSA | Omnipure | 2390 | |
| Streptavidin-coated glass microscope slides | Greiner Bio-one | 439003-25 | |
| polypropylene 384 well plate | Greiner Bio-one | 784201 | |
| Biotin-fluorescein | Sigma | 53608 | |
| contact microarray printer | Aushon | 2470 | Aushon 2470 Microarray Printer |
| contact microarray printer | Gene Machines | OmniGrid 100 | OmniGrid Microarray Printer |
| PBS | Invitrogen | 14190 | |
| Blocking Buffer | ArrayIt | SBB | |
| Hydrophobic wax pen | Vector Labs | H-4000 | ImmEdge Hydrophobic Barrier PAP Pen |
| Silicon Gasket | Grace Bio-labs | 622511 | |
| Hybridization Vessel | Thermo Scientific | 267061 | or similar vessel |
| Fluorescent-dye conjugated secondary antibody | Life Technologies | A-21244 | Alexa Fluor 647 (anti-rabbit) |
| Fluorescent-dye conjugated secondary antibody | Life Technologies | A-21235 | Alexa Fluor 647 (anti-mouse) |
| Wax Imprinter | ArrayIt | MSI48 | |
| Tween-20 | Omnipure | 9490 | |
| Microarray Scanner | Innopsys | InnoScan 1100AL | or equivalent microarray scanner |
| EipTitan Histone Peptide Microarray | Epicypher | 112001 | |
| AbSurance Pro Histone Peptide Microarray | Millipore | 16668 | |
| MODified Histone Peptide Array | Active Motif | 13001 | |
| Histone Code Peptide Microarrays | JPT | His_MA_01 | |
| Wax | Royal Oak | GulfWax | for wax imprinter |
| Humidified Microarray Slide Hybridization Chamber | VWR | 97000-284 | |
| High throughput microscope slide washing chamber | ArrayIt | HTW | |
| Microscope slide centrifuge | VWR | 93000-204 | |
| Antibody 1 | Abcam | 8898 | |
| Antibody 2 | Millipore | 07-473 | |
| Biotinylated histone peptide | EpiCypher | 12-2001 | Example peptide. Similar peptides with various modifications are available from several commercial sources. |
| ImageMagick | https://www.imagemagick.org/script/index.php | ||
| ArrayNinja | https://rothbartlab.vai.org/tools/ |
Etiketler
Bu Makaleden Alıntı Yapın
A Peptide Microarray Technology for Specificity Profiling of Antibodies. J. Vis. Exp. (Pending Publication), e22115, doi: (2024).