The proteins were separated by SDS-PAGE using standard protocols and transferred onto a 0.2 µm nitrocellulose membrane (GE Healthcare) using a Semi-dry blotting apparatus. Protein transfer was performed at 15 V for 20 min in transfer buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS, 20% (v/v) methanol). After blocking non-specific binding with 5% nonfat milk, the blots were probed either with anti-aIF5A antiserum (used at a 1:10,000 dilution in Tris buffered saline solution containing 0.05% Tween 20 and 5% nonfat milk) or with the anti-hypusine antibody (Millipore). The primary antibodies were detected by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell Signaling Technology), employing the enhanced chemiluminescent reagent (SuperSignal West Pico PLUS, Thermo Scientific). The images were visualized with a BioRad ChemiDoc™ MP Imaging system.
Anti hypusine antibody
Manufactured by Merck Group
The Anti-hypusine antibody is a laboratory reagent used in the detection and quantification of the hypusine modification. Hypusine is a unique amino acid found in the eukaryotic translation initiation factor 5A (eIF5A), which plays a crucial role in protein synthesis. This antibody can be utilized in various analytical techniques, such as Western blotting and immunohistochemistry, to study the presence and levels of hypusine-containing proteins.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase hrp conjugated anti rabbit igg, by Cell Signaling Technology (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
0.2 m nitrocellulose membrane, by GE Healthcare (1 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
C digit blot scanner, by LI COR (1 mentions)
Enhanced chemiluminescent reagent, by Euroclone (1 mentions)
0.2 μm nitrocellulose membrane, by GE Healthcare (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
3 protocols using anti hypusine antibody
1
Western Blot Analysis of aIF5A and Hypusine
2
Immunoblot Analysis of eIF5A and Hypusine
3
Western Blot Analysis of aIF5A and Hypusine
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Western blot analysis was performed as previously described (Bassani et al., 2018 (link)) with some modifications. Briefly, 15 μg of total proteins for each sample was separated on SDS-15% polyacrylamide gel using standard protocols and then transferred onto a 0.2-μm nitrocellulose membrane (GE Healthcare) using wet transfer blotting apparatus. Protein transfer was performed at 100 V for 30 min in a transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). Nonspecific binding was blocked using 5% nonfat milk. The membranes were probed overnight at 4°C, either with anti-aIF5A (used at a 1:5,000 dilution in TBS-Tween containing 5% of nonfat milk) or with anti-hypusine antibody (Millipore). The detection of primary antibodies was obtained by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell Signaling Technology), using the enhanced chemiluminescent reagent (EuroClone). The images were visualized with a BioRad ChemiDoc™ MP Imaging System. The quantification of the signals was obtained by the ImageLab™ software (Biorad).
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