Approximately 30 testes from the transgenic line w-; sfGFP-△N-term-Arp53D and the line w-; Arp53D KO; sfGFP-Arp53D (full length) were dissected separately in PBS and centrifuged. After the supernatant was removed, the pellets of testes were flash frozen. Once thawed for immunoblot analysis, 20 µL of 4X NuPAGE LDS sample buffer (Thermo Fisher) was added to each pellet, which was resuspended and boiled for 5 min at 100°C. Protein samples were loaded on a mini-protean TGX stain-free protein gel (BioRad), run with Tris/Glycine/SDS buffer and transferred to a PVDF trans-blot turbo membrane (BioRad). After blocking with 5% milk in Tris-buffered saline (TBS) and 0.1% Tween-20 (TBST), the membrane was probed with anti-GFP and anti-tubulin in TBST for 1 hr at room temperature, followed by three 10 min washes with TBS. The membrane was then incubated for 45 min at room temperature with IR dye 680 anti-chicken (LI-COR) and/or IR dye 800 anti-rabbit 800 nm (LI-COR) in TBST (see Table 4 for dilutions). After three final washes with TBS, the membrane was scanned with 680 nm and 800 nm.
Ir dye 680 anti chicken
Manufactured by LI COR
The IR dye 680 anti-chicken is a near-infrared fluorescent dye designed for use in various life science applications. It is a conjugated molecule that binds specifically to chicken-derived proteins or antibodies. The dye has an excitation wavelength of approximately 680 nanometers and emits fluorescence in the near-infrared region of the electromagnetic spectrum.
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2 protocols using ir dye 680 anti chicken
1
Immunoblotting Analysis of Testis Proteins
2
Immunoblotting of Drosophila Testes
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Approximately 30 testes from the transgenic line w -; sfGFP-△N-term-Arp53D and the line w -; Arp53D KO; sfGFP-Arp53D (full length) were dissected separately in PBS and centrifuged. After the supernatant was removed, the pellets of testes were flash frozen. Once thawed for immunoblot analysis, 20 µL of 4X NuPAGE LDS sample buffer (Thermo Fisher) was added to each pellet, which was resuspended and boiled for 5 min at 100°C. Protein samples were loaded on a mini-protean TGX stain-free protein gel (BioRad), run with Tris/Glycine/SDS buffer and transferred to a PVDF trans-blot turbo membrane (BioRad). After blocking with 5% milk in Tris-buffered saline (TBS) and 0.1% Tween-20 (TBST), the membrane was probed with anti-GFP and anti-tubulin in TBST for 1 hr at room temperature, followed by three 10 min washes with TBS. The membrane was then incubated for 45 min at room temperature with IR dye 680 anti-chicken (LI-COR) and/or IR dye 800 anti-rabbit 800 nm (LI-COR) in TBST (See Table S4 for dilutions). After 3 final washes with TBS, the membrane was scanned with 680 nm and 800 nm.
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