For extraction of protein, K562 pellets were resuspended in RIPA buffer with cOmplete Mini Protease Inhibitor Cocktail (Sigma-Aldrich). Lysates were incubated on ice for 10 minutes and then centrifuged at 16,000 × g for 10 minutes in a pre-chilled centrifuge to remove debris and quantification was carried out with BCA protein assay. Equal amounts of protein (25–50 μg) were loaded onto NuPAGE® Novex 4–12% Bis-Tris Gel 1.5 mm, 10 Well (Invitrogen) and subsequently transferred to nitrocellulose membranes using iBlot™ 2 Transfer Stacks (Invitrogen) for 7 minutes at 14 V in iBlot™ 2 Dry Blotting System (Invitrogen). Western blotting was performed with antibodies against ATF4 (Cell Signaling Technology, #11815), β-actin (Sigma-Aldrich, #A5441), IRDye® 680RD Donkey anti-Rabbit secondary antibody (Li-Cor, #925–68073), and IRDye® 800CW Goat anti-Mouse secondary antibody (Li-Cor, #925–32210) and visualized using the Odyssey® Imaging System.
Irdye 680rd donkey anti rabbit secondary antibody
Manufactured by LI COR
The IRDye® 680RD Donkey anti-Rabbit secondary antibody is a fluorescently labeled antibody product designed for detection and quantification of rabbit primary antibodies in Western blotting, immunocytochemistry, and other applications. The antibody is conjugated with the IRDye 680RD fluorescent dye, which has excitation and emission maxima of 682 nm and 701 nm, respectively.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (2 mentions)
Iblot 2 transfer stack, by Thermo Fisher Scientific (1 mentions)
Iblot 2 dry blotting system, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw goat anti mouse secondary antibody, by LI COR (1 mentions)
Complete mini protease inhibitor cocktail, by Merck Group (1 mentions)
Cholesterol cholesterol ester glo assay kit, by Promega (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
2 protocols using irdye 680rd donkey anti rabbit secondary antibody
1
Protein Extraction and Western Blot Analysis
2
Isolating and Quantifying Membrane-Bound Proteins
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Human monocytes were lysed in RIPA buffer to extract the whole cell lysate or lysed following the two-step membrane-bound protein isolation protocol74 (link) for CH25H detection. The lysates were subjected to western blot analysis. Briefly, blotted membrane was blocked with 5% BSA (Sigma) containing PBS for 1 h, incubated with primary antibodies (Abs) overnight, and incubated with secondary Abs in blocking buffer. β-actin (Sigma, clone AC-15) and Na, K-ATPase 1 protein (CST, #3010 S) was served as loading control, respectively. ATF3 and CH25H were detected using anti-ATF3 Ab (CST, clone: D2Y5W) and anti-CH25H Ab (Invitrogen, #PA5-72329), respectively, and IRDye 680RD donkey anti-rabbit secondary antibody (LI-COR Bioscience). Results were analyzed using a LI-COR CL Odyssey imager.
For analyses of cholesterol and 25HC levels, DCs were isolated from spleen of indicated mice and cell lysate was made using lysis buffer from Promega Cholesterol/Cholesterol Ester-Glo assay kit (#J3190) or by multiple thaw-freeze cycles. ELISA-based analyses of the levels of cholesterol or 25HC was carried out using Cholesterol/Cholesterol Ester-Glo assay ELISA kit (Promega, #J3190) or Mouse 25-hydroxycholesterol ELISA kit (MyBioSource, # MBS7256104) according to the manufacturers’ recommendations.
For analyses of cholesterol and 25HC levels, DCs were isolated from spleen of indicated mice and cell lysate was made using lysis buffer from Promega Cholesterol/Cholesterol Ester-Glo assay kit (#J3190) or by multiple thaw-freeze cycles. ELISA-based analyses of the levels of cholesterol or 25HC was carried out using Cholesterol/Cholesterol Ester-Glo assay ELISA kit (Promega, #J3190) or Mouse 25-hydroxycholesterol ELISA kit (MyBioSource, # MBS7256104) according to the manufacturers’ recommendations.
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