Gel logic 100 digital imaging system

Cell culture was harvested and cell pellet was snap frozen with liquid nitrogen. Total RNA was extracted using TRIzol^® Reagent (Ambion, Austin, TX, USA) and purified with QIAGEN RNeasy Mini Kit in accordance with the manufacturer’s protocol. Genomic DNA contamination was eliminated from total RNA sample using RQ1 RNase-Free DNase kit (Promega, Madison, WI, USA), and RNA purity and quantity were checked with the NanoPhotometer^® P360 device (Implen, Munich, Germany). A total of 500 ng of total RNA was converted to first-strand cDNA with oligo(dT) primer using GoScript^TM Reverse Transcription System (Promega, Madison, WI, USA) in a 20 µL reaction. The resulting cDNA was used as template for real-time RT-PCR using iQ™ SYBR^® Green Supermix (Bio-Rad, Hercules, CA, USA) with the following primers: mCherry_F (5′-ACATCAAGCTGGACATCACC-3′) and mCherry_R (5′-CTTGTACAGCTCGTCCATGC-3′). A total of 10 µL of each RT-PCR product was resolved by electrophoresis on a 1% (w/v) agarose gel, and the gel image was captured with a Kodak Gel Logic 100 Digital Imaging System (Kodak, Rochester, NY, USA).

SDS-PAGE and Lectin Blots used 4–20% Tris-HCl Ready Gels (Bio-Rad). Antibody samples were mixed 1:1 with Laemmli Sample Buffer (Bio-Rad) with a loading concentration of 1% SDS, heated to 95°C for 2 minutes and electrophoresis performed with Tris-Glycine-SDS (0.1% SDS) running buffer on a MiniProtean II (BioRad). Kaleidescope prestained standards (BioRad 161–0324) were run in the end lanes. Standard SDS-PAGE gels were imaged used Coomassie Blue staining, followed by fixing and destain steps. For Lectin Blotting, proteins were transferred to Westran S PVDF membranes (Whatman) using an iBlot system (Invitrogen). The membrane was blocked using Carbohydrate-free blocking buffer (Vector Labs) followed by overnight incubation at 4°C with biotinylated SNA (100 μg/ml). After washing, the membrane was incubated for 1 hour at room temperature with Qdot 625 streptavidin conjugate (Invitrogen, A10196, 1:1000 dilution of 1 μM stock). Bands were visualized and imaged with a Gel Logic 100 Digital Imaging System (Kodak) using a Dark Reader Transilluminator (Clare Chemical).