Odyssey blocking buffer tbs

Cells were homogenized by scraping into 90 µl Urea/SDS buffer (10 mM Tris‐HCl pH 6.8, 6.7 M urea, 1% w/v SDS, 10% v/v glycerol and 7.4 µM bromophenol blue, containing 50 µM phenylmethysulfonyl fluoride and 50 µM N‐methylmaleimide, all Sigma Aldrich). Lysates were sonicated and 10% v/v β‐mercaptoethanol (Sigma Aldrich) added. Proteins were separated by SDS‐PAGE using 10% polyacrylamide gels; 1.5 M Tris, 0.4% w/v SDS, 10% acrylamide/bis‐acrylamide (all Sigma Aldrich), electrophoresis buffer; 25 mM tris‐HCl, 190 mM glycine, 0.1% w/v SDS, pH 8.5 (all Sigma Aldrich). Proteins were transferred to a nitrocellulose membrane using the TurboBlot^™ system (BioRad) and blocked at room temperature in Odyssey® TBS‐Blocking Buffer (Li‐Cor BioSciences) for 1 h. The membranes were probed overnight at 4°C diluted in blocking buffer, washed 3 × 5 min in PBS with 0.1% Tween (both Sigma Aldrich) and probed for 1 h at room temperature in the dark with IRDye® conjugated secondary Abs against goat IgG (800 CW) and rabbit IgG (680 CW), raised in goat or donkey (LiCor BioSciences), diluted in Odyssey® TBS‐Blocking Buffer at 0.05 µg/ml. Membranes were then washed for 3 × 5 min in PBS with 0.1% Tween, rinsed with ddH₂O and imaged using the Odyssey® CLX 9120 infrared imaging system (LiCor BioSciences). Image Studio Light v.5.2 was used to process scanned membranes.

Lysates used for immunoblotting were prepared in ice-cold RIPA Buffer containing HALT protease inhibitors (ThermoFisher). Clarified cell lysates were resolved on 4–15% mini-PROTEAN TGX pre-cast gels (Bio-Rad), and transferred to nitrocellulose membranes using a TransBlot Turbo (Bio-Rad, Hercules, CA, USA). Membranes were blocked with Odyssey TBS Blocking Buffer (Li-Cor, Licoln, NE, USA) and then probed with the appropriate antibodies. Primary antibodies were diluted in TBS + 1% PVP as follows: anti-PDX1, ab47267 1:5000 (Abcam, Cambridge, MA, USA); anti-PDX1, sc-14664, 1:5000 (Santa Cruz, Dallas, TX, USA) anti-Tubulin, T5326, 1:20000 (Sigma); anti-βactin, A5316, 1:5000 (Sigma); anti-eGFP, ab290, 1:5000 (Abcam); anti-HA, ab9110, 1:5000 (Abcam); anti-mCherry, ab125096, 1:2000 (Abcam) anti-OLLAS; NBP1-06713, 1:1000 (Novus, New York, NY); anti-FLAG, F3165-.2MG, 1:400 (Sigma); anti-His, 12698, 1:1000 (CST, Danvers, MA); anti-myc, 2278, 1:1000 (CST), anti-V5, MA5-15253, 1:1000 (ThermoFisher). Membranes were incubated with appropriate antibodies overnight at 4°C. Secondary antibodies (Li-Cor) were diluted 1:10 000 in Odyssey TBS Blocking Buffer (Li-Cor) with 0.2% Tween 20 added and incubated with membranes for 1 h at room temperature. Immunoblots were developed using a Li-Cor Odyssey CLx.

Following fixation, cells were incubated with 1X TBS-1% Triton X-100 to permeabilize for 15 min. Cells were then incubated with TBS Odyssey Blocking Buffer (LI-COR) at RT for 1.5 h while rocking. Cells were stained with primary antibodies as indicated: PAB597 (1:40), ab34756 (1:1000), ERK1/2 (1:500), or pERK1/2 (1:500) in TBS Odyssey Blocking Buffer (LI-COR) at 4°C overnight while rocking. After primary incubation, cells were washed with 1X TBS-T and incubated with either an anti-mouse or anti-rabbit LI-COR 800 secondary antibody (1:10,000) and CellTag 700 (1:500) at RT for 1 h while rocking. Secondary-alone wells were treated only with species-appropriate LI-COR 800 secondary antibody (1:10,000). Cells were washed with 1X TBS-T three times and aspirated to remove all liquid prior to scanning. Using a LI-COR Odyssey CLx Infrared Imaging system, plates were immediately scanned to detect 700 and 800 nm channel intensities. Plates were read at a 42 μm resolution, at medium quality, with a 3.0 mm focus offset. After scanning, 700 and 800 nm channels were aligned using the Image Studio software (version 5.2) equipped with the ICW module. After scanning, the ICW analysis grid (Image Studio) was applied to the plate image to outline each well and images were then processed using Image J (NIH).