LFQ_TurboID-STING_anti-biotin_antibody

The STING-null RAW264.7 cells expressing TurboID-STING were treated with DMSO or 30 μg/ml DMXAA, and 500 µM biotin was added to the medium. After 1-h incubation, the cells were lysed in 6 M guanidine-HCl containing 100 mM HEPES-NaOH (pH 7.5), 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication, and centrifuged at 20,000 g for 15 min at 4ºC. The supernatants were recovered, and proteins were purified by methanol/chloroform precipitation and solubilized by 0.1% RapiGest SF in 50 mM triethylammonium bicarbonate. After repeated sonication and vortex, the proteins were digested with trypsin at 37ºC overnight. The resultant peptide solutions were captured on anti-biotin (A7C2A) rabbit monoclonal antibody-conjugated beads in the presence of 1 mg/ml Pefabloc SC during 3 h of incubation at 4ºC. After washing with TBS four times, and with ultrapure water twice, the biotinylated peptides were eluted with 100 µL of 0.2% TFA and 80% ACN twice. The combined eluates were desalted using GL-Tip SDB, evaporated in a SpeedVac concentrator, and redissolved in 0.1% TFA and 3% ACN.

将表达TurboID-STING的STING基因缺陷型RAW264.7细胞用二甲基亚砜（DMSO）或30 μg/ml的DMXAA处理，并向培养基中加入500 μM生物素。孵育1小时后，将细胞在含有100 mM羟乙基哌嗪乙硫磺酸-氢氧化钠（HEPES-NaOH，pH 7.5）、10 mM三(2-羧乙基)膦（TCEP）以及40 mM氯乙酰胺（CAA）的6 M盐酸胍溶液中裂解。裂解液经加热与超声处理使其充分溶解，随后在4℃下以20000 g离心15分钟。收集上清液，通过甲醇-氯仿沉淀法纯化蛋白质，再用含0.1% RapiGest SF的50 mM碳酸氢三乙胺溶液溶解蛋白。经反复超声和涡旋混匀后，将蛋白质置于37℃下用胰蛋白酶酶解过夜。所得肽段溶液在1 mg/ml Pefabloc SC存在的条件下，于4℃孵育3小时，使生物素化肽段与偶联抗生物素（A7C2A）兔单克隆抗体的磁珠结合。依次用Tris缓冲盐溶液（TBS）洗涤四次、超纯水洗涤两次后，使用100 μL的0.2%三氟乙酸（TFA）与80%乙腈（ACN）的混合液洗脱生物素化肽段，重复洗脱操作两次。合并所有洗脱液，使用GL-Tip SDB固相萃取柱进行脱盐处理，经真空离心浓缩仪（SpeedVac concentrator）蒸发浓缩后，用0.1%三氟乙酸与3%乙腈的混合液复溶。