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CDK4 Polyclonal Antibody

  • polyclonal antibody-Elabscience
  • polyclonal antibody-Elabscience
  • polyclonal antibody-Elabscience
  • polyclonal antibody-Elabscience
  • polyclonal antibody-Elabscience
  • polyclonal antibody-Elabscience
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  • polyclonal antibody-Elabscience
  • polyclonal antibody-Elabscience
  • polyclonal antibody-Elabscience
  • polyclonal antibody-Elabscience
  • polyclonal antibody-Elabscience
  • polyclonal antibody-Elabscience
  • polyclonal antibody-Elabscience
  • polyclonal antibody-Elabscience
  • polyclonal antibody-Elabscience
  • polyclonal antibody-Elabscience
  • polyclonal antibody-Elabscience
  • polyclonal antibody-Elabscience
    产品货号:E-AB-40286

    别称:Cdk 4, cdk4, CDK4 protein, CDK4_HUMAN, Cell division kinase 4, Cell division protein kinase 4, CMM 3, CMM3, Crk3, Cyclin dependent kinase 4, Cyclin-dependent kinase 4, Melanoma cutaneous malignant 3, MGC14458, p34 cdk4, PSK J3, PSK-J3

    规格:
    • 20μL
    • 60μL
    • 120μL
    • 200μL
    数量:
    - +
    价格: ¥400

    宿主: Rabbit

    反应性: H,M,R

    应用: WB,IHC

    货期: 现货+顺丰时间

    加入购物车 比较 咨询 说明书

    Overview

    Synonyms Cdk 4, cdk4, CDK4 protein, CDK4_HUMAN, Cell division kinase 4, Cell division protein kinase 4, CMM 3, CMM3, Crk3, Cyclin dependent kinase 4, Cyclin-dependent kinase 4, Melanoma cutaneous malignant 3, MGC14458, p34 cdk4, PSK J3, PSK-J3
    Swissprot P11802
    Source Rabbit
    Reactivity Human,Mouse,Rat
    Immunogen Recombinant Human Cyclin-dependent kinase 4 protein(PKSH033817)
    Application WB(Detection kit: E-IR-R304),IHC(Detection kit: E-IR-R217)
    Recommended dilution WB 1:1000-1:2000 IHC 1:100-1:300
    Concentration 1mg/mL
    Clonality Polyclonal

    Operation Manual

    CDK4 Polyclonal Antibody Western Blot

    操作指南(下载PDF

    为了方便操作及WB结果的准确性,现提供了WB配套试剂盒(Cat# E-IR-R304),含有从样本制备到抗体检测用到的试剂。请根据具体需要订购合适的试剂盒。

    实验流程 实验条件
    胶浓度 15%
    转膜 150mA,1h
    封闭 5%脱脂奶粉,2h
    一抗 1:1000
    二抗 1:5000

    更多详细实验条件和操作步骤,请点击.. 详情 ↓

    蛋白样本的制备

    1. 样本的处理

    1)组织样本处理:
    a.取待测组织样本,用预冷的PBS(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和10μL原矾酸钠进行匀浆裂解,均浆后冰上震荡裂解30min。
    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),超声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,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30min。
    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),超声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,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℃。

    注:待测样本建议总蛋白上样量为50μg,尽量保持各待测样本上样体积接近10μL。

    电泳

    1.根据靶蛋白的分子量的大小,配制15%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),以验证目的分子量大小及转膜程度。加入1x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,直至电泳结束。

    转膜(湿转)

    1.根据靶蛋白的分子量选择0.22μm孔径的PVDF膜(Cat# E-BC-R329)。PVDF膜先在甲醇中浸泡1min使其活化,然后将PVDF膜浸泡于1x转膜缓冲液(Cat# E-BC-R333)中(含20%甲醇),同时将滤纸和纤维垫也浸泡在转膜缓冲液中待用。

    2.按照黑色板(负极)-纤维垫-滤纸-凝胶-PVDF膜-滤纸-纤维垫-白色板(正极)依次放好,排出气泡,夹紧后放入湿转电转槽内。建议转膜条件为150mA,1h。请务必保证转膜过程在低温条件下进行。

    注:此为湿转的参考步骤,如用其他转膜方法,请依具体情况进行调整。


    免疫印迹

    1.用含5% 脱脂奶粉的TBST(Cat# E-BC-R335)(封闭液)浸泡PVDF膜,室温摇床封闭2h

    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1:1000稀释CDK4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3次,15min每次

    4.用含2%脱脂奶粉的TBST溶液按照说明书推荐的稀释比选取1:5000稀释Goat Anti-Rabbit IgG(H+L)(peroxidase/HRP conjugated)(Cat# E-AB-1003),室温摇床孵育1h。

    5.TBST充分洗涤PVDF膜3次,15min每次

    显色曝光

    1.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。

    2.PVDF膜从TBST洗液中取出后用滤纸吸干水分,均匀滴加ECL混合液于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

    Properties

    Cellular localization Cytoplasm, Nucleus, Membrane 
    Tissue specificity Highest expression level in right ovary
    Isotype IgG
    Purification Antigen Affinity Purification
    Conjugation Unconjugated
    Storage instructions Store at -20℃. Avoid freeze / thaw cycles.
    Storage buffer PBS with 0.05% Proclin300, 50% glycerol, pH7.3.
    Background The protein encoded by this gene is a member of the Ser/Thr protein kinase family. This protein is highly similar to the gene products of S. cerevisiae cdc28 and S. pombe cdc2. It is a catalytic subunit of the protein kinase complex that is important for cell cycle G1 phase progression. The activity of this kinase is restricted to the G1-S phase, which is controlled by the regulatory subunits D-type cyclins and CDK inhibitor p16(INK4a). This kinase was shown to be responsible for the phosphorylation of retinoblastoma gene product (Rb). Mutations in this gene as well as in its related proteins including D-type cyclins, p16(INK4a) and Rb were all found to be associated with tumorigenesis of a variety of cancers. Multiple polyadenylation sites of this gene have been reported.

    Images

    • Western Blot analysis of NIH/3T3, K562 cells, Rat spleen and Rat stomach using CDK4 Polyclonal Antibody at dilution of 1:1000

    • Western Blot analysis of 293T cells using CDK4 Polyclonal Antibody at dilution of 1:1000

    • Western Blot analysis of HepG2 cells using CDK4 Polyclonal Antibody at dilution of 1:1000

    • Immunohistochemistry of paraffin-embedded Human lung cancer using CDK4 Polyclonal Antibody at dilution of 1:200

    • Immunohistochemistry of paraffin-embedded Human liver cancer using CDK4 Polyclonal Antibody at dilution of 1:100

    • Immunohistochemistry of paraffin-embedded Human kidney using CDK4 Polyclonal Antibody at dilution of 1:100

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