gp120 shedding induced by MβCD alone (no PT present) was measured using Western blots as described previously.4 (link) Briefly, MβCD was mixed with virus stocks (1:1, v/v) and incubated at 37 °C for 30 min before being spun for 2 h at 21130 × g at 4 °C. Sample supernatants were mixed 1:1 with Laemmli buffer and boiled at 95 °C for 5 min before being loaded onto 10% SDS-PAGE gels. After gel electrophoresis, the protein was transferred onto a 0.45 μm PVDF membrane (Immobilon-P, Millipore), blocked with 5% milk solubilized in PBS containing 0.1% Tween-20 and then stained with sheep anti-gp120 (D7324, Aalto), followed by donkey antisheep HRP (Invitrogen) antibodies. Luminol substrate (Advansta) was added and the protein bands exposed on chemiluminescence film (Blue Ultra, Genemate) and developed (M35-A X-OMAT processor, Kodak). The developed film was then scanned, and the corresponding image was analyzed with ImageJ (NIH) densitometry.
Donkey antisheep hrp
Manufactured by Thermo Fisher Scientific
Donkey anti-sheep HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is used for the detection of sheep-derived primary antibodies in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p, by Merck Group (1 mentions)
M35 a x omat processor, by Kodak (1 mentions)
Ab214428, by Abcam (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Af2557, by R&D Systems (1 mentions)
Mini protean tgx stain free gels 4 20, by Bio-Rad (1 mentions)
Transblot turbo mini size pvdf membrane, by Bio-Rad (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
2 protocols using donkey antisheep hrp
1
Quantification of gp120 Shedding by MβCD
2
Western Blot Analysis of Lysosomal Proteins
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Proteins were resolved by Mini-Protean TGX stain-free gels 4–20% (BioRad 4568093) and transferred to TransBlot Turbo mini-size PVDF membrane (BioRad). Membranes were fixed with 0.4% paraformaldehyde (PFA) then rinsed three times with ultrapure water using the Milli-Q EQ 7000 Ultrapure Water Purification System. The membrane was blocked in 5% powdered milk in 1X tris-buffered saline with Tween-20 (TBS-T), and primary antibodies and secondary antibodies were dissolved in this same buffer. Information on the antibodies used can be found in Table 1. Primary antibodies used were LAMP1 (rabbit, Abcam ab24170) diluted 1:1000, PGRN (sheep, R&D AF2557) diluted 1:400 and Cathepsin B (CTS B)(rabbit, Abcam ab214428) diluted 1:1000. Secondary antibodies Goat anti Rabbit HRP (Jackson Immunoresearch 111-035-144) and Donkey anti-Sheep HRP (Invitrogen A16041) were diluted 1:2000. The membrane was developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo scientific 34577) and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo scientific 34096) and visualized on the Li-cor Odyssey FX system.
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