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Histone H3 Monoclonal Antibody

  • Cat.No.:E-AB-22003

  • 宿主: Mouse
  • 反应性: H,M,R,Yeast
  • 应用: WB,IHC-p,IF,IP

订购:E-AB-22003

规格:
  • 20μL
  • 60μL
  • 120μL
价格: ¥580
数量:

Test Application

  • Verified Samples

    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.

    IHC
    (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.

    IF
    (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.

  • Dilution

    WB 1:500-1:2000, IHC 1:50-300, IF 1:100-500, IP 1:100-1:300

蛋白样本的制备

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℃。

电泳

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。

转膜

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。

免疫印迹

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 .

显色曝光

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。

附录

Product Details

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 Details

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

Background

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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