Cat.No.:E-AB-22003
Reactivity | Application |
Human | WB (Hela,) ![]() Western Blot analysis of Hela cells, RAW264.7 cells, Mouse brain and Rat brain using Histone H3 Monoclonal Antibody at dilution of 1:5000. (uterus,) ![]() Immunohistochemistry of paraffin-embedded Human uterus tissue using Histone H3 Monoclonal Antibody at dilution of 1:200. |
Rat |
WB (brain,) ![]() Western Blot analysis of Hela cells, RAW264.7 cells, Mouse brain and Rat brain using Histone H3 Monoclonal Antibody at dilution of 1:5000. (liver,) ![]() Immunofluorescence analysis of Rat liver tissue using Histone H3 Monoclonal Antibody at dilution of 1:200. |
Mouse |
WB (RAW264.7,brain,) ![]() ![]() Western Blot analysis of Hela cells, RAW264.7 cells, Mouse brain and Rat brain using Histone H3 Monoclonal Antibody at dilution of 1:5000. Western Blot analysis of Hela cells, RAW264.7 cells, Mouse brain and Rat brain using Histone H3 Monoclonal Antibody at dilution of 1:5000. |
WB 1:500-1:2000, IHC 1:50-300, IF 1:100-500, IP 1:100-1:300
1.样本的处理
1)组织样本的处理:
a) 取待测组织样本,用预冷的PBS(0.01 M, pH=7.4)(Cat# E-BC-R187) 充分洗涤,洗去组织表面血液及内部杂物。
b) 称重剪碎,加入适量比例的RIPA裂解液混合物 (Cat# E-BC-R327)(1mL的RIPA裂解液中加入10μL PMSF(Cat# E-BC-R287)和10μL原矾酸钠(Cat# E-BC-R250)进行匀浆裂解。建议按组织重量:RIPA体积=3:10的比例匀浆,例如0.3g的组织样本对应1mL的RIPA裂解液,具体体积可根据实验需要适当调整。
c) 在35~40%的功率下,超声处理样本1min(冰浴条件下进行),每次2s,间隔2s,确保细胞充分裂解,降低样本粘度。
d) 4℃下12,000rpm离心10min。
e) 取上清,待测定蛋白浓度。
2)细胞样本处理:
a) 收集待测细胞样本,用预冷的PBS(0.01 M, pH=7.4)充分洗涤,洗去培养基等成分(一般建议洗涤3次)。
b) 加入适量比例的RIPA裂解液混合物(1mL RIPA裂解液中加入10μL PMSF和10μL原矾酸钠)进行冰上裂解。建议6孔板中每孔加入0.1mL RIPA裂解液(细胞中的蛋白含量可能会不同,可适当调整加入裂解液的体积)。
c) 在35~40%的功率下,超声处理样本1min(冰浴条件下进行),每次2s,间隔2s,确保细胞充分裂解,降低样本粘度。
d) 4℃下12,000rpm离心10min。
e) 取上清,待测定蛋白浓度。
2.BCA法测定蛋白浓度(参见BCA蛋白浓度测定试剂盒(Cat# E-BC-K318)说明书)。
3. 用PBS调整蛋白浓度,按照蛋白样本:5×SDS上样缓冲液=4:1的比例加入5×SDS上样缓冲液(Cat# E-BC-R288),沸水煮10min。12,000rpm离心2min,收取上清。变性后的蛋白即可进行后续的Western Blot实验,或保存于 -20℃或-80℃。
1.根据靶蛋白的分子量的大小,配制 0% 的分离胶。每个泳道加入待测样本,并预留一个泳道加入5μL的预染蛋白Marker (Cat# E-BC-R273),以验证转膜程度及目的分子量大小。加入电泳缓冲液 ( Cat# E-BC-R331),开始电泳。
2.80V恒压电泳,待溴酚蓝指示剂移动至浓缩胶与分离胶交界处成线状,改为恒压120v,跑完全程。
1.根据靶蛋白的分子量选择 μm 孔径的PVDF膜(Cat# E-BC-R266)。PVDF膜先在甲醇中浸泡1min使其活化,然后将PVDF膜浸泡于转膜缓冲液(Cat# E-BC-R333)中。同时将滤纸和纤维垫也浸泡在转膜缓冲液中待用。
2.遵照转膜仪生产商的说明进行湿转、半干转或干式转印。
1.用含5% 脱脂奶粉(Cat# E-BC-R337)的TBST(Cat# E-BC-R335)(封闭液)浸泡PVDF膜,室温摇床封闭。
2.用含5% 脱脂奶粉的TBST溶液按照说明书推荐的稀释比选取稀释IMP3抗体,使PVDF膜浸泡于一抗孵育液中,4℃孵育过夜,并不断轻轻晃动。
3.TBST充分洗涤PVDF膜 .
4.用含2%脱脂奶粉的TBST溶液(配比见附录)按照说明书推荐的稀释比选取稀释Goat Anti-Rabbit IgG(H+L)(peroxidase/HRP conjugated)(Cat# E-AB-1003),室温摇床孵育1 h。
5.TBST充分洗涤PVDF膜 .
1.PVDF膜从TBST洗液中取出后用滤纸吸干水分。
2.将ECL化学发光检测试剂盒(Cat# E-BC-R347)中的A液与B液按1:1比例混匀,均匀滴加于PVDF膜上,排出气泡,即可曝光。
3.调节对比度,多次曝光获取最佳图片效果。
Clonality | Monoclonal |
Isotype | IgG |
Concentration | 1mg/mL |
Storage | Store at -20℃. Avoid freeze / thaw cycles. |
Buffer | PBS with 0.02% sodium azide, 50% glycerol, pH7.4 |
Purification Method | Protein A purification |
Research Areas | Cancer, Epigenetics and Nuclear Signaling |
Clone No. | Clone:5B1 |
Conjugation | Unconjugated |
Immunogen | Recombinant Protein |
Abbre | Histone H3 |
Synonyms | HIST1H3A,H3FA,HIST1H3B,H3FL,HIST1H3C,H3FC,HIST1H3D,H3FB,HIST1H3E,H3FD,HIST1H3F,H3FI,HIST1H3G,H3FH,HIST1H3H,H3FK,HIST1H3I,H3FF,HIST1H3J,H3FJ,Histone H3.1,Histone H3/a,Histone H3/b,Histone H3/c,Histone H3/d,Histone H3/f,Histone H3/ |
Swissprot | P68431,Q71DI3,P84243 |
Calculated MW | 15kDa |
Observed MW | 15kDa |
Cellular Localization | Extracellular region or secreted,extracellular exosome,extracellular region,Nucleus,nuclear chromosome,nuclear chromosome, telomeric region,nuclear nucleosome,nucleoplasm,nucleus,Other locations:nucleosome,protein complex |
Application | Products |
WB | Western Blot Detection Kit(E-IR-R304) |
IHC | 2-step plus Poly-HRP Anti Mouse/Rabbit IgG Detection System (with DAB solution)(E-IR-R217) |
Core component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling. |
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