Serine/threonine-protein kinase that acts as a regulator of the p38/MAPK14 stress-activated MAPK cascade and of the MAPK8/JNK cascade. In response to DNA damage, involved in the G2/M transition DNA damage checkpoint by activating the p38/MAPK14 stress-activated MAPK cascade, probably by mediating phosphorylation of upstream MAP2K3 and MAP2K6 kinases. Inhibits basal activity of the MAPK8/JNK cascade and diminishes its activation in response to epidermal growth factor (EGF). Positively regulates canonical T cell receptor (TCR) signaling by preventing early PTPN6/SHP1-mediated inactivation of LCK, ensuring sustained TCR signaling that is required for optimal activation and differentiation of T cells (PubMed:30373850). Phosphorylates PTPN6/SHP1 on 'Thr-394', leading to its polyubiquitination and subsequent proteasomal degradation (PubMed:38166031). Required for cell surface expression of metalloprotease ADAM10 on type 1 transitional B cells which is necessary for their NOTCH-mediated development into marginal zone B cells (By similarity). Also required for the NOTCH-mediated terminal differentiation of splenic conventional type 2 dendritic cells (By similarity). Positively regulates osteoblast differentiation by acting as an upstream activator of the JNK pathway (PubMed:32807497). Promotes JNK signaling in hepatocytes and positively regulates hepatocyte lipid storage by inhibiting beta-oxidation and triacylglycerol secretion while enhancing lipid synthesis (PubMed:34634521). Restricts age-associated inflammation by negatively regulating differentiation of macrophages and their production of pro-inflammatory cytokines (By similarity). Plays a role in negatively regulating the abundance of regulatory T cells in white adipose tissue (By similarity) Serine/threonine-protein kinase that acts as a regulator of the p38/MAPK14 stress-activated MAPK cascade and of the MAPK8/JNK cascade (By similarity). In response to DNA damage, involved in the G2/M transition DNA damage checkpoint by activating the p38/MAPK14 stress-activated MAPK cascade, probably by mediating phosphorylation of upstream MAP2K3 and MAP2K6 kinases (By similarity). Inhibits basal activity of the MAPK8/JNK cascade and diminishes its activation in response to epidermal growth factor (EGF) (By similarity). Positively regulates canonical T cell receptor (TCR) signaling by preventing early PTPN6/SHP1-mediated inactivation of LCK, ensuring sustained TCR signaling that is required for optimal activation and differentiation of T cells (By similarity). Phosphorylates PTPN6/SHP1 on 'Thr-394', leading to its polyubiquitination and subsequent proteasomal degradation (By similarity). Required for cell surface expression of metalloprotease ADAM10 on type 1 transitional B cells which is necessary for their NOTCH-mediated development into marginal zone B cells (PubMed:28068307). Also required for the NOTCH-mediated terminal differentiation of splenic conventional type 2 dendritic cells (PubMed:33214146). Positively regulates osteoblast differentiation by acting as an upstream activator of the JNK pathway (PubMed:32807497). Promotes JNK signaling in hepatocytes and positively regulates hepatocyte lipid storage by inhibiting beta-oxidation and triacylglycerol secretion while enhancing lipid synthesis (By similarity). Restricts age-associated inflammation by negatively regulating differentiation of macrophages and their production of pro-inflammatory cytokines (PubMed:37400834). Plays a role in negatively regulating the abundance of regulatory T cells in white adipose tissue (PubMed:37347461) Serine/threonine-protein kinase that acts as a regulator of the p38/MAPK14 stress-activated MAPK cascade and of the MAPK8/JNK cascade. In response to DNA damage, involved in the G2/M transition DNA damage checkpoint by activating the p38/MAPK14 stress-activated MAPK cascade, probably by mediating phosphorylation of upstream MAP2K3 and MAP2K6 kinases. Inhibits basal activity of the MAPK8/JNK cascade and diminishes its activation in response to epidermal growth factor (EGF). Positively regulates canonical T cell receptor (TCR) signaling by preventing early PTPN6/SHP1-mediated inactivation of LCK, ensuring sustained TCR signaling that is required for optimal activation and differentiation of T cells. Phosphorylates PTPN6/SHP1 on 'Thr-396', leading to its polyubiquitination and subsequent proteasomal degradation. Required for cell surface expression of metalloprotease ADAM10 on type 1 transitional B cells which is necessary for their NOTCH-mediated development into marginal zone B cells. Also required for the NOTCH-mediated terminal differentiation of splenic conventional type 2 dendritic cells. Positively regulates osteoblast differentiation by acting as an upstream activator of the JNK pathway. Promotes JNK signaling in hepatocytes and positively regulates hepatocyte lipid storage by inhibiting beta-oxidation and triacylglycerol secretion while enhancing lipid synthesis. Restricts age-associated inflammation by negatively regulating differentiation of macrophages and their production of pro-inflammatory cytokines. Plays a role in negatively regulating the abundance of regulatory T cells in white adipose tissue
a. 贴壁培养细胞收集
去除贴壁细胞的培养液，用PBS、NS或无血清培养基清洗1次，低速离心，弃上清，留取沉淀。
b. 悬浮培养细胞收集
速离心悬浮细胞，弃上清，收集沉淀。手指轻弹细胞，使其松散。
c. 组织样本收集
把组织剪切成细小的碎片，越小越好。取液氮或超低温冰箱中冷冻30min以上的组织，迅速用液氮研磨，研磨过程尽量控制在1~2min之内，以减少蛋白的降解。
（2）总蛋白提取
a. 细胞/组织裂解
将装有细胞沉淀或组织碎片的容器完全插入冰中。细胞沉淀按照1mL裂解液/107个细胞(1个T75培养瓶细胞量)的比例加入相应体积的裂解液（细胞量足够时都加入3mL，不足时根据细胞量计算），裂解20min，每隔5min将离心管置于涡旋振荡仪上震荡10s。组织碎片按照0.5mL 裂解液/100mg组织向匀浆器中加入蛋白裂解液，每3min研磨一次，重复5次，使组织尽量碾碎。（裂解液中根据需要选择添加或不添加蛋白酶抑制剂）。
b. 离心
把裂解好的样品配平后，置于预冷的高速冷冻离心机中，12000 rpm，15min。
c. 蛋白变性
完成离心后，上清即为蛋白提取液。吸取少量蛋白提取液做蛋白浓度测定。向剩余的蛋白提取液的离心管中加入1/5上清体积的5×Loading Buffer（最终工作液为1X），待干式恒温器温度升至95℃后，将1.5mL离心管插入加热孔中，95℃加热变性10min，待液体完全冷却后置于-20℃保存。
（3）蛋白浓度测定（BCA法）
a. BCA工作液的配置
根据样品数量，按50体积BCA试剂A加入1体积BCA试剂B（50:1）配置适量BCA工作液，充分混匀。BCA工作液室温24h内稳定。
b. 标准品测定
取10μl蛋白标准品（5mg/ml BSA）稀释至50μl，使终浓度为1mg/ml。稀释后的蛋白标准品可以-20℃长期保存。此标准品溶液的稀释液可使用去离子水或1*PBS。将标准品按0、1、2、4、8、12、16、20μl加入到96孔板中，加稀释液补足到20μl（见附表）。加适当体积样品到96孔板的样品孔中，如果样本不足20μl，需加稀释液补足到20μl。请注意记录样品体积。各孔加入200μl BCA工作液，37℃放置20-30min。用酶标仪测定A562，或540-595nm之间的其他波长吸光度。根据标准曲线和使用的样品体积计算出样品的蛋白浓度。
a. Western Transfer Buffer至少提前2h （即开始电泳后）放入-20℃冰箱预冷，但注意避免结冰。
b. 根据胶体大小,将Filter Paper及Nitrocellulose membrane剪裁至合适尺寸。
c. 目的蛋白＞20KD选择0.45μm NC膜/PVDF膜；目的蛋白＜20KD选择0.2μmNC膜或PVDF膜，选择完毕后将NC膜放在Western Transfer Buffer中浸泡备用，注意如使用的是PVDF膜需先放入甲醇中浸泡5-10min，再放入Western Transfer Buffer中浸泡备用。
（2）裂解液&洗杂液：Cell lysis buffer for IP (without inhibitors)
（3）蛋白酶抑制剂
（4）封闭液：含 3% BSA 的 1X PBS
（5）1×PBS 缓冲液
（6）5×loding buffer（使用时用去离子水稀释至工作浓度即可）
（7）Control IgG （AC005/ AC011/AC034）
二、实验步骤
1、样本处理
（1）贴壁培养细胞
a. 取裂解液室温溶解混匀，根据需要选择添加或不添加蛋白酶抑制剂。
b. 去除贴壁细胞的培养液，用PBS、NS或无血清培养基清洗1次，低速离心，弃上清，留取沉淀。
c. 按照6孔板每孔加入100~200μl裂解液的比例，加入裂解液。移液器轻轻吹打，使裂解液和细胞充分接触。通常裂解液作用于细胞1~5s内，细胞会被裂解。
d. 1000~12000g，离心3~5min（如果用冷冻离心机4℃效果更佳），取上清。
（2）悬浮培养细胞
a. 取裂解液室温溶解混匀，根据需要选择添加或不添加蛋白酶抑制剂。
b. 速离心悬浮细胞，弃上清，收集沉淀。
c. 手指轻弹细胞，使其松散。按照6孔板每孔加入100~200μl裂解液的比例，加入NP-40裂解液。通常6孔板每孔加入100~200μl裂解液已经足够，但如果细胞密度非常高可以适当加大裂解液的用量150~200μl，再用手指轻弹以充分裂解细胞。充分裂解后应无明显沉淀。
d. 1000~12000g，离心3~5min（如果用冷冻离心机4℃效果更佳），取上清。
（3）组织样本
a. 取裂解液室温溶解混匀，根据需要选择添加或不添加蛋白酶抑制剂。
b. 把组织剪切成细小的碎片，越小越好。
c. 取液氮或超低温冰箱中冷冻30min以上的组织，迅速用液氮研磨，研磨过程尽量控制在1~2min之内，以减少蛋白的降解。
d. 按照每20mg组织加入100~200μl裂解液的比例，加入含有PMSF的裂解液。冰上或4℃裂解30-60min。（步骤3、4也可采用以下过程：按照每20mg组织加入100~200μl裂解液的比例加入NP-40裂解液。用玻璃匀浆器或组织研磨器匀浆，直至充分裂解，过程尽量控制在1~2min之内，以减少蛋白的降解。）
e. 按照每20mg组织加入100~200μl裂解液的比例，加入裂解液。
f. 1000~12000g，4℃离心10~15min（如无低温离心机，室温下离心也可），取上清。
2、磁珠预处理
（1）将rProtein A/G Plus MaqPoly Beads颠倒或漩涡混匀，翻转瓶身发现底部无黑色沉淀即可。
（2）取30μl rProtein A/G Plus MaqPoly Beads至新的EP管中，放在磁分离器上，待溶液澄清后，用移液器吸弃保护液。
（3）将EP管从磁分离器上取下来，加入1ml Cell lysis buffer for IP (without inhibitors)，混匀，放置在磁分离器上，收集磁珠，用移液器吸弃洗杂液，重复2次。
