Image studio light

Media was removed from one well of a 6-well plate (cells at ~90% confluency) for each cell line and cells were washed gently with PBS. Cells were resuspended in PBS via gentle resuspension and placed into a microcentrifuge tube. Cells were then pelleted at 600 g for 3 min. PBS was removed and 250 μL of 95°C 2% SDS was added to lyse cells. The mixture was vortexed and heated at 95°C for 5 min. Fifty microliters of 6x sample buffer containing 5% β-mercaptoethanol was added and vortexed. Samples were stored at −20°C until use.
For western blot analysis 10–15 μL lysates were added to wells a 4–15% gradient gel. The gel was then run at 180 V until the dye reached the bottom. Proteins were transferred to a nitrocellulose membrane. The membrane was stained with Ponceau S Stain and a picture was taken. The membrane was then washed and blocked for 20 min. Primary antibodies in blocking buffer were added and the membrane was incubated overnight at 4°C. The following day the membrane was washed 3x and incubated with secondary antibody in 5% evaporated milk in PBS for 40 min. Membrane was washed 4x and imaged using the Odyssey imaging system. Analysis was done in Licor Image Studio light.

A serial dilution of MBP fused to FLAG®-, HA-, myc- and ALFA-tags was prepared in PBS pH7.4, 0.1 µg mL⁻¹ BSA. In total 1 µL of each dilution was spotted on nitrocellulose membranes. Membranes were blocked and washed with 5% milk powder in TBS-T. Established monoclonal antibodies (anti-FLAG® M2, Sigma #F1804; anti-myc 9E10 Synaptic Systems #343 011; anti-HA F-7, SantaCruz #sc-7392) were used in combination with a secondary goat anti-Mouse IgG IRDye800CW (Li-COR #925–32210, dilution 1:500) to detect FLAG®-, myc- and HA-tag, respectively. The ALFA-tag was detected using a FluoTag®-X2 anti-ALFA nanobody (NanoTag Biotechnologies #N1502) directly coupled to IRDye800CW. All primary antibodies and the nanobody were used at 2.7 nM final concentration. Detection of MBP by a rabbit polyclonal serum recognizing MBP (Synaptic Systems, dilution 1:500) and an anti-rabbit IgG IRDye680RD (Li-COR #925–68071, dilution 1:5000) served as an internal loading control. Membranes were scanned using Odyssey CLx (Li-COR). Quantifications were performed using ImageStudioLight (Li-COR).

Cells were lysed by sonication in 20 mM TEA, 4% SDS, 1 mM EDTA buffer. Samples were separated in a Bolt 4–12% Bis-Tris Plus gel using Bolt MES SDS Running Buffer (Invitrogen) following the manufacturer’s instructions. Separated proteins were electro-transferred onto an Immobilon-P Membrane, PVDF (Merck Millipore). Primary antibody binding was detected by incubation with a peroxidase-conjugated secondary antibody and chemiluminescent substrate Luminata Forte (Merck Millipore). Carbonylated proteins were detected and analyzed following derivatization of protein carbonyl groups with 2,4-dinitrophenylhydrazine, using the OxyBlot kit (Merck Millipore). Immunodetection was performed utilizing 10 µg of protein per lane with a primary antibody directed against dinitrophenylhydrazone. Equal loading was demonstrated by using the same amount of each of the samples, separated by SDS PAGE in the same conditions and stained with sensitive Coomassie Blue stain [21] (link). Density analysis was performed using Image Studio Light (Li-Cor). The optical density for each lane was normalised to the averaged total density of all lanes on the gel and expressed as percentage of the optical density.