Anti his tag antibody

HEK293 cells were transfected with pcDNA3.1/His vector alone or containing Ikba and pFLAG-CMV-1 vector alone or containing Redd1, Redd1^KKAA, or Redd1^KKRAAA using Lipofectamine 2000. Cells were rinsed two times with ice-cold phosphate-buffered saline and lysed in modified RIPA buffer [100 mM Tris-HCl (pH 7.6), 5 mM EDTA, 50 mM NaCl, 50 mM β-glycerophosphate, 50 mM NaF, 0.1 mM Na₃VO₄, 1% Brij-35, 0.5% sodium deoxycholate, and 1 mM phenylmethylsulphonyl fluoride] for 30 min on ice. After centrifugation for 15 min at 12,000 × g at 4 °C, the supernatant was collected. Total protein (1 mg) was precleared with protein G plus/protein A-agarose (Millipore, 50% slurry in modified RIPA buffer) for 1 h at 4 °C. The indicated antibodies (1–3 μg) were added to 500 μL of precleared protein extracts. The precleared protein extracts (500 μL) were mixed with 2 μg of anti-FLAG M2 antibody (#F3165, Sigma-Aldrich) or anti-His-Tag antibody (#2365, Cell Signaling) and rotated for 24 h at 4 °C, as described^{18 (link)}. Then, 30 μL of protein A/G plus agarose beads (50% slurry) were added to the mixture, which was rotated at 4 °C for 2 h. Immunoprecipitates were collected by centrifugation and washed with modified RIPA buffer three times. Samples were boiled for 10 min with a loading buffer. Immune complexes were separated using SDS-PAGE (10–15%) and subjected to immunoblot analysis.

Proteins were separated by SDS-PAGE and transferred to a PVDF membrane by dry transfer using the iBlot dry blotting system from Invitrogen (Waltham, MA, USA). Membranes were blocked for 1 h at room temperature in 5% w/v milk or BSA in TBS-T (20 mM Tris-HCl pH 8.0, 137 mM NaCl, 0.05% Tween), followed by incubation with primary antibody overnight at 4 °C. Primary antibodies: anti-His-tag antibody (#A00186, GenScript, 1:5000) (Piscataway, NJ, USA), anti-His-tag antibody (#12698, Cell Signaling, 1:1000) (Danvers, MA, USA), anti-NEIL2 antibody (#124106, Abcam, 1:500) (Cambridge, UK), anti-P35 antibody (anti-p35, SantaCruz, #sc-518009, 1:2500) (Dallas, TX, USA), and anti-actin antibody (#A2228, Sigma, 1:10,000) (Merck, Darmstadt, Germany). Subsequently, the membrane was washed in TBS-T and incubated with secondary anti-mouse IgG Horseradish Peroxidase Linked (GE Healthcare, #NA931, 1:5000) (Chicago, IL, USA) or anti-rabbit IgG Horseradish Peroxidase Linked (GE Healthcare, #NA934, 1:5000) (Chicago, IL, USA) antibody for 1 h at room temperature, followed by additional washing in TBS-T and detection with ECL Prime Western Blotting Detection Reagent from GE Healthcare (Chicago, IL, USA) according to the manufacturer’s protocol.

Glycan array slides were printed on SuperEpoxy 3 (Arrayit) activated substrates using an Arrayit Spotbot Extreme contact printer as previously described^{28 (link)}. For each subarray 2 μg of SubB proteins were pre-complexed with anti-His tag antibody (Cell signalling) and Alexa555 secondary and tertiary antibodies (rabbit anti-mouse; goat anti-rabbit) at a ratio of 2:1:0.5:0.25 in a final volume of 500 μL. This 500 μL antibody protein complex was added to a 65 μL gene frame (Thermo Scientific) without a coverslip. Washing and analysis was performed as previously described^{27 (link)}.