Protein a g sepharose

Collected cells were extracted for 30 min in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1% Triton X-100) containing protease inhibitor cocktail (Sigma-Aldrich, MO, USA). After centrifuge at 2,0000 g for 30 min at 4 °C, supernantant was pre-cleared by incubation with Protein A/G-Sepharose (Sigma-Aldrich, MO, USA). Pre-cleared supernatants were incubated with transgelin-2 antibody or control IgG for 3 h at 4 °C. Then Protein A/G Sepharose was added to pull down the immune-complex. After washing, the immunoprecipiated proteins were analyzed by SDS-PAGE.

HEK293 cells or A549 cells were co-transfected with the indicated plasmids with or without virus infection for 24 h. The transfected cells were then harvested and lysed in NP-40 lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 1 mM EDTA with protease inhibitor cocktails]. For each immunoprecipitation, 1 ml of lysate was incubated for 4 h at 4°C with 0.5 μg of the indicated antibody or control IgG and 30 μL of protein A/G-Sepharose (Sigma). The beads were washed three times with 1 ml of lysis buffer containing 500 mM NaCl. The precipitates were analyzed by using standard immunoblotting procedures.

As previously reported20 (link), aliquots of 1000 μg of protein samples in 1 mL of lysis buffer from each treatment were pre-cleared by incubation with 30 μL of protein A/G Sepharose (Sigma) for 2 hours at 4 °C rotation. The pre-cleared samples were incubated with the specific anti-SIN1 antibody (1 μg/mL) overnight at 4 °C rotation. 20–30 μL of protein A/G Sepharose were added to the samples 2 hours at 4 °C rotation. The beads were washed and boiled, followed by Western blot assay.

The HSP70.PC was purified by immunoprecipitation with rabbit anti-human HSP70 mAb (Abcam) as described previously[20 (link)]. Briefly, DC-tumor fusion cells and tumor cells were collected and washed three times with ice cold phosphate-buffered saline (PBS, pH 7.4). Then cells were incubated in lysis buffer containing a protease inhibitor cocktail (Roche) (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1% (v/v) Nonidet P-40, 1 mM NaV) on ice for 30 min, then centrifuged at 13,000 rpm for 15 min. The mAb against HSP70 (Abcam, Burlingame, California) was added to the supernatant at a concentration of 10 μg/ml and the mixture was gently rotated at 4°C overnight. Then, 80 μl protein A/G-sepharose (1:1) (Sigma-Aldrich) was added and incubated at 4°C for an additional 90 min. The protein sepharose was collected by centrifugation and after extensive washing in lysis buffer, the immunoprecipitates were eluted into high salt PBS (500 mM NaCl). The concentration of protein in the eluate was determined for use in lymphocyte stimulation.

Cells (1 × 10⁷) were lysed in immunoprecipitation buffer (50 mmol/liter HEPES, pH 7.6, 150 mmol/liter NaCl, 5 mmol/liter EDTA, 0.1% Nonidet P-40). After centrifugation (10 min at 15,000 × g) to remove particulate material, the supernatant was incubated with each antibody (2 μg/ml) with constant agitation at 4°C. The immune complexes were precipitated with protein A/G-Sepharose (Sigma) and analyzed by Western blot. To examine the effect of GTGKT peptide on the binding of CAGE to GSK3β, biotin-GTGKT peptide (Peptron, Daejeon, Korea) was transfected with Lipofectamin and PlusTM reagent (Invitrogen, San Diego, CA). After incubation for 48 h, whole-cell extracts were incubated with anti-biotin antibody (2 μg/ml) for 12 h at 4°C and immune complexes were precipitated with streptavidin-linked agarose beads for 30 min at 4°C. After five washes with lysis buffer, the bound proteins were eluted by boiling in 2X Laemli SDS loading buffer and were then subjected to SDS-PAGE followed by Western blotting analysis with anti-CAGE antibody.