Odyssey blocking buffer in tbs

Rats were anesthetized with 2–3% isoflurane, the kidneys were excised, decapsulated and cut longitudinally and dissected into three sections (cortex, outer and inner medulla), snap frozen in liquid nitrogen, and stored at −80°C. The cortex was used for determination of BMAL1 protein abundance. Cortex samples were homogenized using a nuclear isolation kit (Cayman Chemical Company, Ann Arbor, MI). Proteins from the nuclear compartment (20 μg) were used along with positive and negative controls taken from renal cortex of wildtype and Bmal1 knockout mice. Nuclear extracts were separated by a 15% SDS-PAGE and transferred to PVDF membranes. Non-specific protein binding was blocked by a 1 h incubation in blocking buffer (Odyssey® Blocking buffer in TBS, Li-Cor, Lincoln). Bmal1 was detected by a primary rabbit polyclonal antibody (Signalway Antibody LLC, Baltimore, MD, catalog # 21415) with a donkey anti-goat IgG-Alexa Fluor 680 (0.2 μg/ml, A32860, ThermoFisher) by the Odyssey CLx (Li-Cor). Specificity was confirmed in an identical blot with the exception of incubating the primary antibody with the antigenic peptide (catalog # 61415, Signalway Antibody LLC).

HEK293T cells in 6-well plates were transfected with 2.8 μg 3×HA-tagged TRM editor and 1.2 μg non-targeting guide RNA. After 48 h, cells were washed with PBS and lysed in 200 μl RIPA buffer (Thermo Fisher) with PMSF and cOmplete Protease Inhibitor Cocktail (Roche). Lysate was denatured at 95 °C, 1 μl per sample was loaded into a 10-well NuPAGE 4-12% Bis-Tris gel (Thermo Fisher), and gel was dry-transferred to a 0.2 μm PVDF (polyvinylidene difluoride) membrane (Thermo Fisher) using an iBlot 2 Dry Blotting System (Thermo Fisher) for 7 min at 20 V. Membranes were stained with mouse anti-HA (1:1000, Cell Signaling Technology 2367) and rabbit anti-Histone H3 (1:2500, Abcam ab1791) in Odyssey Blocking Buffer in TBS (LI-COR) overnight at 4 °C. After washing 3× with TBST (TBS + 0.5% Tween-20), membranes were incubated with IRDye-labeled secondary antibodies goat anti-mouse 680RD (LI-COR 926-68070) and donkey anti-rabbit 800CW (LI-COR 926-32213) diluted 1:5000 in Odyssey Blocking Buffer for 1 h at room temperature. The membrane was finally washed 3× with TBST, then imaged on an Odyssey Imaging System (LI-COR).