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  • NF-kappa B signaling pathway

     

    Pathway description:
    NF-Kappa B (Nuclear Factor-Kappa B) is a heterodimeric protein composed of different combinations of members of the Rel family of transcription factors. The Rel/ NF-Kappa B family of transcription factors are involved mainly in stress-induced, immune, and inflammatory responses. In addition, these molecules play important roles during the development of certain hemopoietic cells, keratinocytes, and lymphoid organ structures. NF-Kappa B is also an important regulator in cell fate decision, such as programmed cell death and proliferation controlled, and is critical in tumorigenesis. NF-Kappa B is composed of homo- and heterodimers of five members of the Rel family including NF-Kappa B1, NF-Kappa B2,RelA,RelB,and c-Rel. NF-Kappa B can be activated by exposure of cells to LPS or inflammatory cytokines such as TNF or IL-1,growth factors, lymphokines, oxidant-free radical, inhaled particles, viral infection or expression of certain viral or bacterial gene products, UV irradiation, B or T-cell activation, and by other physiological and non physiological stimulin.
     
    Selected Reviews:
    Baldwin AS Jr. (1996) The NF-Kappa B and I kappa B proteins:new discoveries and insights.Annu Rev Immunol.14,649. 
    Vigo Heissmeyer and Daniel Krappmann,et al. (2001)Shared Pathways of I B Kinase-Induced SCF TrCP-Mediated Ubiquitination and Degradation for the NF-B Precursor precursor precursor p105 and I Bacterial. Molecular and Cellular Biology.21,1024.
    Joel L,Pomerantz and David Baltimore.(2002)Two pathway to NF-Kappa B.Molecular Cell.10,693.
  • WB常见问题分析

     WB简介

    蛋白印迹是通过聚丙烯酰胺凝胶电泳,将不同分子量大小的蛋白分离并转移到杂交膜上,再通过一抗/二抗复合物对蛋白质进行检测的一种免疫生化技术。

     WB基本操作

    提取标本蛋白并定量

    凝胶电泳分离蛋白

    转移蛋白质到杂交膜

    收膜和封闭

    孵育一抗

    洗涤

    孵育酶联物(二抗或者亲和素-HRPAP

    洗涤

    底物显色或曝光检测

    结果分析

     

    1.高背景

    可能原因

    验证或解决办法

    膜污染

    使用干净镊子,戴手套操作,避免污染

    膜干燥

    保证充分的反应液,避免出现干膜现象

    封闭不完全

    封闭液不合适

    延长封闭时间

    比较尝试不同封闭液

    缓冲液污染

    使用新配置的缓冲液

    漂洗不完全

    增加漂洗时间和缓冲液体积

    抗体浓度过高

    降低一抗、二抗浓度

    抗体与阻断蛋白有交叉反应

    选择无交叉反应的封闭液,洗涤液中加入Tween-20可以减少交叉反应

     

    2.信号弱或无信号

    可能原因

    验证或解决办法

    抗原量不足

    增加上样量

    蛋白质降解

    重新制备样品

    抗原被封闭液遮挡

    优化封闭液,缩短封闭时间,减小封闭液中的蛋白浓度

    样本中不含靶蛋白或靶蛋白含量太低

    设置阳性对照。若靶蛋白含量低,可适当增加样本上样量

    膜的错误选择

    选择合适孔径的膜。>22KD 0.45um

                                  <22KD 0.22um

    转膜不完全

     

    转膜后确定转膜效率,保证胶与膜充分结合,保证电极正确装配,控制转膜温度,优化转膜电流及时间

    甲醇浓度过高

    过高的甲醇浓度会导致蛋白质与SDS分离,从而沉淀在凝胶中。同时会使凝胶收缩或变硬,抑制高分子蛋白的转移。因此,要根据不同分子量选择合适的甲醇浓度

    洗膜过度

    缩短洗涤时间或减少洗涤次数

    封闭过度

    减少封闭剂的量或缩短封闭时间。更换不同封闭剂类型

    一抗失效

    选择在有效期内的抗体,抗体避免反复冻融。选择现配现用的工作液

    一抗反应不充分

    增加抗体浓度,延长孵育时间

    试剂之间不匹配

    一抗与组织种属,一抗与二抗或和底物与酶系统之间不匹配。通过设置内参可以验证二级检测系统的有效性

    HRP 抑制

    所有溶液和容器中避免含有叠氮化钠

    酶和底物失效

    直接将酶和底物进行混合,如果不显色说明酶失活了。选择在有效期内、有活性的酶联物,使用新鲜底物

    曝光时间过短

    延长曝光时间

     

     3.非特异性条带

    可能原因

    验证或解决办法

    蛋白上样量过大

    降低样本上样量

    蛋白降解

    使用新鲜制备的样本,并使用蛋白抑制剂

    细胞传代过多,导致蛋白变异

    使用原代或传代少的细胞做对照

    抗体浓度过高

    降低抗体浓度

    交叉反应

    选择单克隆抗体或亲和纯化的抗体,保证抗体特异性

    不同异构体的存在

    有些来源于同一基因的蛋白有不同异构体,每个异构体蛋白大小都是不同的

    二抗非特异性结合

    增加二抗对照,选择其他二抗

    底物太灵敏

    选择合适底物

    曝光时间过长

    减少曝光时间

     

    4.   条带位置不对

    可能原因

    验证或解决办法

    二聚体或多聚体的存在

    增加蛋白质变性过程及强度

    蛋白修饰

    如糖基化、磷酸化等修饰状态会导致蛋白分子量增加

     

     5.   条带内出现不显影圆点

    可能原因

    验证或解决办法

    转膜过程有气泡

    转膜时赶尽气泡

     

     6.   条带不完整

    可能原因

    验证或解决办法

    底物孵育不均匀

    均匀孵育底物

     

     7.   微笑条带

    可能原因

    验证或解决办法

    电泳速度过快

    减少电压等减慢电泳速度

    电泳温度过高

    在冷室或冰浴中进行电泳

     

     8.   反影(白色条带,黑色背景)

    可能原因

    验证或解决办法

    HRP浓度过高

    降低二抗浓度

     

     9.   背景有黑色斑点

    可能原因

    验证或解决办法

    转膜时有气泡或抗体分布不均匀

    尽量去除气泡,抗体孵育时保持摇动

    封闭剂中有聚集体

    使用前过滤封闭试剂

    抗体与封闭剂反应

    选择合适的封闭剂

    HRP耦联二抗中有聚集体

    过滤二抗试剂,去除聚集体

     

     10.Markera变黑

    可能原因

    验证或解决办法

    抗体与marker蛋白反应

    marker和样本之间空出一个孔不上样

     

    WB实验的主要试剂

    1WB实验膜

    PVDF膜,硝酸纤维素膜或尼龙膜。

    2.封闭试剂

    BSA或脱脂奶粉。

    3.蛋白Marker

    主要目的是确定靶蛋白的分子量大小;使用预染Marker还可以实时检测电泳分离情况并可以转移到膜上。

     4.蛋白提取及定量

    类别

    品名

    SAB对应货号

     

    蛋白提取

    总蛋白提取试剂盒

    PE001

    核蛋白提取试剂盒

    PE002

    膜蛋白提取试剂盒

    PE003

     

                         蛋白定量

    BCA 蛋白定量试剂盒

    PA001

    Bradford 蛋白定量试剂盒

    PA002

     5.一抗

    *选择适合检测标本种属的一抗(说明书上有验证信息)

    *选择适用于WB实验方法的一抗(说明书上有验证信息)

    *选择单克隆抗体或经过亲和纯化的多克隆抗体

    *根据说明书推荐的浓度优化最佳的抗体稀释比

    6.内参

    内参的作用:

    1).检测整个WB实验过程及体系是否正常工作。

    2).半定量的标准。

    内参的选择:

    一般为稳定表达的管家基因蛋白,来源最好选择和同时使用的一抗来源相同的,以方便二抗的选择。

    内参选择: 

    MW(kDa) 全细胞 线粒体 细胞核 细胞膜
       Vinculin(124kDa)    

    NakATPase(112kDa)  

       

     

    Lamin B1(66kDa) 

     
         HSP60(60kDa)    
      α-tubulin(55kDa)      
       β-tubulin(50kDa)      
       β-actin(43kDa)      
       GAPDH(37kDa)    TBP(38kDa)  
       

     VDAC1(30kDa)

     PCNA(29kDa)  
       Cofilin(20kDa)  COXIV(20kDa)    
         

     HistoneH3(15kDa)

     

     

     7.二抗

    1).二抗的选择:

    根据一抗来源种属及抗体lg亚型选择合适的二抗。

    2).二抗的使用:

    根据说明书推荐的浓度稀释二抗。

    孵育条件一般为室温12小时。

     常用二抗

    二抗名称

    SAB对应货号

    Goat anti-Rabbit lgG Secondary antibody HRP conjugated

    L3012

    Goat anti-Mouse IgG Secondary antibody HRP conjugated

    L3032

    Rabbit anti-Goat IgG Secondary antibody HRP conjugated

    L3042

    Goat anti-Rabbit lgG,FITC conjugated

    L3202

    Goat anti-Mouse lgG,FITC conjugated

    L3302

     8.底物

    A.显色底物:操作简便,灵敏度低,如TMBDABBCIPNBT

    B.化学发光底物:灵敏度高,需要曝光检测设备(暗室、曝光设备和胶片)或者凝胶成像设备。

    9.各种溶液

    A.封闭液:一般为含有15BSA或者5%脱脂奶粉的PBSPBST0.1MPh7.4)或TBSTBST;也可以购买商业化的试剂。

    B.稀释液:一般为封闭液或者单独的PBSTTBST,也可以购买商业化的试剂。

    C.洗涤液:一般为PBSTTBST,也可以购买商业化的试剂。

  • Alzheimer pathway

     

  • ELISA实验步骤

     样本处理方法:

    组织样本:切割样本后,称取重量。加入一定量预冷的 PBS ,缓冲液中可加入 1 μg/L 蛋白酶抑制剂或50 U/mL的Aprotinin( 抑肽酶)。用手工或匀浆器将样本匀浆充分。1000 ×g离心20 分钟左右。收集上清并分装,置于-20℃或-70℃保存。如有必要,可以将样品浓缩干燥。分装后一份待检测,其余冷冻备用。没有针对所有样本类型的最佳方法,客户参考报道文献的处理方法进行样本处理

    细胞培养上清:检测分泌性的成份时,用无菌管收集。1000×g离心20分钟左右。收集上清。检测细胞内的成份时,用 PBS稀释细胞悬液,细胞浓度达到 100万 /mL左右。通过反复冻融,以使细胞膜破坏并释放出细胞内成份。1000×g离心 20 分钟左右。收集上清。保存过程  中如有沉淀形成,应再次离心。

    血清:全血标本请于室温放置2小时或4℃过夜后于1000 x g离心20分钟,取上清即可检测,或将标本放于-20℃或-80℃保存,但应避免反复冻融。

    血浆:可用EDTA或肝素作为抗凝剂,标本采集后30分钟内于2 - 8° C 1000 x g离心15分钟,或将标本放于-20℃或-80℃保存,但应避免反复冻融。

     

    一、夹心法

     

    1. 加样:分别设空白孔、标准孔、待测样品孔。除空白孔外,余孔分别加标准溶液或待测样品100ul,轻轻混匀,酶标板加上盖,37℃温育120分钟。

    2. 弃去液体,甩干,不用洗涤。直接每孔加检测溶液A工作液 100ul,轻轻晃动混匀,酶标板加上覆膜,,37℃温育60分钟。

    3. 弃去液体,甩干,洗板 3次。每次浸泡1-2分钟,大约350-400μl/每孔,甩干(也可轻拍将孔内液体拍干)。

    4. 每孔加检测溶液B工作液 100ul,酶标板加上覆膜37℃温育60分钟,洗板5次(方法同3)。

    5. 依序每孔加底物溶液90ul,37℃避光显色(时间应严格控制在10-20分钟之间,此时肉眼可见标准品的前3-4孔有明显的梯度蓝色,后3-4孔梯度不明显)。

    6. 依序每孔加终止溶液50ul,终止反应(此时蓝色立转黄色)。终止液的加入顺序应尽量与底物液的加入顺序相同。为了保证实验结果的准确性,底物反应时间到后应尽快加入终止液。

    7. 用酶联仪在450nm波长依序测量各孔的光密度(OD值)。在加终止液后立即进行检测。

    注意:具体操作步骤以产品说明书为准

     

    二、竞争法

    1. 加样:分别设空白孔、标准孔、待测样品孔。空白孔加样品稀释液 50μl,余孔分别加标准品或待测样品50μl

    2. 立即在每个孔中加入检测溶液A工作液 50μl,轻轻晃动混匀,酶标板加上覆膜,37℃温育60分钟。

    3. 弃去液体,甩干,洗板 3次。每次浸泡1-2分钟,大约350-400μl/每孔,甩干(也可轻拍将孔内液体拍干)。

    4 .每孔加检测溶液B工作液100μl,酶标板加上覆膜37℃温育45分钟,洗板5次,方法同3。

    5. 依序每孔加底物溶液90μl,酶标板加上覆膜37℃避光显色

    6. 依序每孔加终止溶液50μl,终止反应,此时蓝色立转黄色。

    7. 用酶联仪在450nm波长依序测量各孔的光密度(OD值)。 在加终止液后立即进行检测。

    注意:具体操作步骤以产品说明书为准

     

    注:

    1.试剂准备:所有试剂都必须在使用前达到室温,使用后请立即按照说明书要求保存试剂。 实验操作中请使用一次性的吸头,避免交叉污染。

    2.加样:加样或加试剂时,请注意在吸取标本 / 标准品,酶结合物或底物时,第一个孔与最后一个孔加样之间的时间间隔如果太大,将会导致不同的 “预孵育”时间,从而明显地影响到测量值的准确性及重复性。一次加样时间(包括标准品及所有样品)最好控制在10分钟内,     如标本数量多,推荐使用多道移液器加样。

    3.孵育:为防止样品蒸发,试验时将反应板放于铺有湿布的密闭盒内,酶标板加上盖或覆膜,以避免液体蒸发;洗板后应尽快进行下步操作,任何时侯都应避免酶标板处于干燥状态;同时应严格遵守给定的孵育时间和温度。

    4.洗涤:洗涤过程中反应孔中残留的洗涤液应在滤纸上充分拍干,勿将滤纸直接放入反应孔中吸水,同时要消除板底残留的液体和手指印,避免影响最后的酶 标仪读数。

    5.试剂配制:试剂在使用前请手甩几下或少时离心处理,以使管壁或瓶盖的液体沉积到管底。标准品、工作液请依据所需的量配置使用,并使用相应的稀释液配制,不能混淆。请精确配置标准品及工作液,尽量不要微量配置(如吸取检测溶液A时,一次不要小于10μl),以     避免由于不准确稀释而造成的浓度误差;请勿重复使用已稀释过的标准品、检测溶液A工作液或检测溶液B工作液。

    6.底物:底物请避光保存,在储存和温育时避免强光直接照射。

    7.建议检测样品时均设双孔测定,以保证检测结果的准确性。

     

    洗板方法

    手工洗板方法:吸去(不可触及板壁)或甩掉酶标板内的液体;在实验台上铺垫几层吸水纸,酶标板朝下用力拍几次;将推荐的洗涤缓冲液至少0.4ml注入孔内,浸泡1-2分钟,根据需要,重复此过程数次。自动洗板:如果有自动洗板机,应在熟练使用后再用到正式实验过程中。

    ELISA标准曲线制作

    可以采用各种绘图软件来绘制ELISA标准曲线,下面以“Curve Expert1.4”软件为例,软件官方链接

     

  • Hedgehog signaling pathway

     

    Hh信号传递受靶细胞膜上两种受体Patched(Ptc)和Smoothened(Smo)的控制。受体Ptc由肿瘤抑制基因Patched编码,是由12个跨膜区的单一肽链构成,能与配体直接结合,对Hh信号起负调控作用。受体Smo由原癌基因Smothened 编码,与G蛋白偶联受体同源,由7个跨膜区的单一肽链构成,N端位于细胞外,C端位于细胞内,跨膜区氨基酸序列高度保守,C 末端的丝氨酸与苏氨酸残基为磷酸化部位,蛋白激酶催化时结合磷酸基团。该蛋白家族成员只有当维持全长时才有转录启动子的功能,启动下游靶基因的转录;当羧 基端被蛋白酶体水解后,就形成转录抑制子,抑制下游靶基因的转录。Smo是Hh信号传递所必须的受体。在无Hh、Ptc的情况下,激活Smo可导 致Hh 靶基因的活化;基因Smo突变时,可出现与Hh 基因突变相同的表征。

  • AMPK signaling pathway

     

               AMPK(AMP-activated protein kinase)信号通路是一种细胞内的信号通路,参与细胞代谢的调节,与细胞的能量代谢密切相关。AMPK是一种重要的蛋白激酶,由蛋白激酶激活蛋白激酶(LKB1)激活,能够响应细胞的能量状态和代谢状态,调节细胞的代谢和生长。AMPK信号通路的主要功能是维持细胞能量平衡,调节葡萄糖和脂肪酸代谢。AMPK活化能够刺激葡萄糖的摄入和运输,促进葡萄糖的利用,同时缺乏能量供应时,AMPK能够促进脂肪酸的氧化,以维持细胞能量平衡。AMPK的活化还能够促进蛋白质的合成,细胞周期的调节,促进细胞的生长和增殖,以维持细胞的稳态。此外,AMPK的活化还与其他相关的信号通路进行调节,如PI3K信号通路、mTOR信号通路、Wnt信号通路等。AMPK的活化可能对治疗糖尿病、肥胖等疾病具有重要的潜在治疗作用。

  • Notch signaling pathway

     

              Notch信号通路由Notch受体、Notch配体(DSL蛋白)、CSL (CBF-1,Suppressor of hairless,Lag的合称)DNA结合蛋白、其他的效应物和Notch的调节分子等组成。1917年,Morgan及其同事在突变的果蝇中发现Notch基因,因该基因的部分功能缺失会在果蝇翅膀的边缘造成缺刻(Notch)而得名。哺乳动物有4种Notch受体(Notch1- 4)和5种Notch配体(Delta-like 1, 3, 4,Jagged1和Jagged2)。Notch信号的产生是通过相邻细胞的Notch配体与受体相互作用,Notch蛋白经过三次剪切,由胞内段(NICD)释放入胞质,并进入细胞核与转录因子CSL结合, 形成NICD/CSL转录激活复合体,从而激活HES、HEY、HERP等碱性-螺旋-环-螺旋(basic-helix-loop- helix,bHLH)转录抑制因子家族的靶基因,发挥生物学作用。

  • B CELL Pathway

     

  • Autophagy Pathway

     

           自噬 Autophagy,或称自体吞噬是一个涉及到细胞自身结构通过溶酶体机制,负责将受损的细胞器、错误折叠的蛋白及其他大分子物质等运送至溶酶体降解并再利用的进化保守过程。自噬是广泛存在于真核细胞的现象,并且可分为巨自噬、微自噬和分子伴侣介导的自噬三大类。这是一个受到紧密调控的步骤,此步骤是细胞生长、发育与稳态中的常规步骤,帮助细胞产物在合成、降解以及接下来的循环中保持一个平衡状态。目前已有多份研究表明自噬在许多细胞的分化进程中被不同程度地激活,例如参与血管生成、成骨分化、脂肪生成、神经发生等过程。自噬效应的发生取决于自噬流过程是否完成,而自噬流的意思是自噬的完整动态过程,包括自噬体形成、自噬体与溶酶体融合及后续内含物的降解和回收。

  • JAK-STAT Pathway

     

               JAK -STAT途径是多种细胞因子和生长因子的主要信号传导机制。JAK激活刺激细胞增殖,分化,细胞迁移和凋亡。这些细胞事件对于造血,免疫发育,乳腺发育和泌乳,脂肪形成,两性性生长和其他过程至关重要。JAK(janus kinase)是一类非受体酪氨酸激酶家族,已发现四个成员,即Jak1 、Jak2 、Jak3 和Tyk2。JAK的N端结构域与受体相结合,C端为激酶结构域。每种激酶成员与特异的细胞因子受体结合。JAK的底物为STAT,即信号转导子和转录激活子(signal transducer and activator of transcription,STAT),N端具有SH2结构域和核定位信号(NLS),中间为DNA结合域,C端有保守的,对其活化至关重要的酪氨酸残基。共发现7个STAT家族成员,分别命名为STAT1至STAT7。STAT被JAK磷酸化后发生二聚化,然后穿过核膜进入核内调节相关基因的表达,这条信号通路称为JAK-STAT途径。

  • mTOR Pathway

     

              mTOR 通路受多种细胞信号的调控,包括有丝分裂生长因子、胰岛素等激素、营养素(氨基酸、葡萄糖)、细胞能量水平和应激条件。PI3K/Akt(v-Akt小鼠胸腺瘤病毒癌基因同源1)信号转导通路是通过mTOR 传递信号的主要通路,在介导细胞存活和增殖中起重要作用。通过 PI3K/Akt 通路的信号是由与细胞膜上的受体结合的生长因子的有丝分裂刺激启动的。这些受体包括IGFR (胰岛素样生长因子受体)、PDGFR (血小板衍生生长因子受体)、EGFR (表皮生长因子受体)和HER 家族。来自激活的受体的信号直接传递到PI3K/Akt 通路,或者,也可以通过由致癌蛋白RAS 激活的生长因子受体激活。RAS 是另一个信号转导的中枢开关,而且已证实是MAPK (丝裂原活化蛋白激酶)信号转导通路的关键激活子。胰岛素也可通过IRS1/2 (胰岛素受体底物-1/2)激活PI3K/Akt 通路。胰岛素结合激活IR (胰岛素受体)酪氨酸激酶,使IRS1 或IRS2 磷酸化。PI3K 通过P85 调节亚基中的SH2 (Src-Homology-2)结构域与磷酸化IR 结合。这种相互作用激活了p110 催化亚基。然后,PI3K 催化膜结合的PIP2 (磷脂酰肌醇(4,5)二磷酸)转化为PIP3 (磷脂酰肌醇(3,4,5)-三磷酸)。PIP3 然后与Akt 的pleckstrin 同源结构域结合,通过二聚化和暴露其催化位点而导致Akt 的激活。

  • T-CELL Pathway

     

           T细胞受体(TCR)在T细胞的功能和免疫突触的形成中起着关键作用。它在T细胞和抗原呈递细胞(APC)之间提供连接。TCRs激活促进了一系列信号级联,最终通过调节细胞因子的产生、细胞存活、增殖和分化来决定细胞的命运。T淋巴细胞的激活是免疫系统有效反应的关键事件。TCR激活受各种共刺激受体调节。CD28在T细胞激活过程中提供了一种必要的共刺激信号,可增加IL-2(白细胞介素-2)的产生,增加T细胞增殖,并防止无能和细胞死亡的诱导。通过B7-1或B7-2连接CD28有助于使T细胞和抗原呈递细胞膜接近。除CD28外,许多其他跨膜受体也调节TCR信号的特定元件,如CD45和CD4。TCR激活的早期事件是淋巴细胞蛋白酪氨酸激酶(Lck)使TCR/CD3复合物胞浆侧的免疫受体酪氨酸基激活基序(ITAM)磷酸化。CD45受体酪氨酸磷酸酶调节Lck和其他Src家族酪氨酸激酶的磷酸化和激活。TCR激活还通过激活ZAP70下游的GTP结合蛋白Rac和PAK导致细胞骨架重排。TCR信号的负调控也很重要,以检查与该通路相关的免疫反应过度激活。SIT(SHP2相互作用跨膜衔接蛋白)是最近发现的一种跨膜衔接蛋白,通过ITIM(免疫受体酪氨酸基抑制基序)与SHP2(含SH2的蛋白酪氨酸磷酸酶-2)相互作用,该复合物是TCR介导信号的关键负调节器。此外,CTLA4(细胞毒性T淋巴细胞抗原-4)也对T细胞活化产生负性调节。跨膜蛋白CTLA4也可作为天然抑制剂。一旦T细胞被激活,无论什么疾病过程使其启动,身体都有一个自然过程来关闭T细胞途径,使其不会失控。

  • ErbB signaling pathway

     

            ERBB2编码的蛋白属于表皮生长因子受体家族,该家族包括HER1(erbB1,EGFR)、HER2(erbB2,NEU)、HER3(erbB3)及HER4(erbB4),也属于受体酪氨酸激酶家族。ERBB2没有与配体结合的结构域,但可以与家族成员形成异源二聚体来结合配体如表皮生长因子(EGF),从而通过磷酸化激活下游蛋白如MAPK和PI3K。ERBB2的过表达在多种肿瘤中被发现,常见的为乳腺癌和卵巢癌。

  • Wnt signaling pathway

     

  • Hippo signaling pathway

     

  • p53 signaling pathway

     

  • 免疫组化疑难解答

     Troubleshooting tips for IHC common problems:

    1. Non-specifc staining

    2. No staining

    3. Weak staining 

    4. Strong Staining 

     

    1.Non-specifc staining

     

    Causes Solutions
    Improper preparation of sections Improve ways of sampling and preparation
    Inadequate deparaffinization of the sections Increase the deparaffinization time
    Tissue contains endogenous peroxidase Use 0.3% v/v fresh H2O2 for blocking and increase the blocking incubation time
    Tissue contains endogenous biotin Use IHC biotin blocking agent
    Blocking of protein may be insufficient Increase the blocking time
    Charge adsorption Block with nonimmune animal serum
    The antibody is not pure Change suitable antibody
    Primary antibody concentration may be too high Try decreasing the antibody concentration
    The sections have dried out Avoid sections being dried out in the process of experiments
    Washes may be insufficient Increase the times of washes and the washing time

     

    Non-specifc staining: Improved:

             

               

    Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue showing cytoplasmic and nuclear staining using NFκB-p65 Phospho-Ser276 Antibody #11011.

     

    2.No staining

    Causes Solutions
    Improper tissue processing Try to improve the condition and sampling again
    No antigen in the tissue Set a positive control to verify the experiment results
    The antibody is not active Don’t use out-of-date antibody kits
    Incompatible secondary and primary antibodies Use secondary antibody that was raised against the species in which the primary was raised
    Incompatible staining system Change compatible staining system
    Improper operation and leave out important steps Follow strict operating procedure and set a positive control

      

    No staining:  Improved:

      

    Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using Histone H3 Di-Methyl-Lys27 Antibody #11583.

     

    3.Weak staining 

    Causes Solutions
    Improper tissue fixation or too high temperature when fixing Use appropriate fixation way or fixation time
    Too high baking slides temperature and too long baking time Choose appropriate temperature and time for baking slides
    The antigen may be damaged Let fresh tissues be fixed in time and for not to exceed 24 hours
    Over blocking of protein Reduce the blocking time
    The antibody has drained away Ensure that the sections are placed in a horizontal position  when incubating
    The antibody concentration may be too low or incubation time may be too short Increase the antibody concentration and incubation time
    The room temperature may be too low. Lower than 15℃. Incubator at 37℃ or increase incubation time
    No draining off buffer solution when adding the reagent results in the reagent being diluted. Do drain off buffer solution but avoid sections being dried out.
    Excessive washing Wash moderately
    Always verify the expiration date of the reagent prior to use Change reagents timely

     

    Weak staining: Improved:

      

    Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using mTOR Phospho-Ser2448 Antibody#11221.

     

    4.Strong Staining

     

    Causes Solutions
    The primary antibody concentration may be too high or incubation time may be too long Reduce the primary antibody concentration or incubation time.
    The incubation temperature may be too high Incubate at 4℃ or at room temperature
    The incubation time of HRP conjugated secondary antibody may be too long Reduce the incubation time
    Inadequate washing Increase the times of washing

     

    Strong Staining: Improved:

     

    Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using FKHR Phospho-Ser256 Antibody#11115.

     

  • 免疫印迹疑难解答

    Troubleshooting tips for western blotting common problems: 
        1.High background
        2.Low or no signal
        3.Non-specific bands
        4.Wrong band location
        5.Invisible dots on the bands
        6.Incomplete bands
        7.Smile effect of the bands
        8.White bands on a black blot 
        9.Black dots on the blot
        10.Marker lane is black

     

    1.High Background

    Causes Solutions
    Membrane fouling Use clean tweezer and operate with gloves to prevent membrane fouling.
    The membrane has dried out Incubate in sufficient reaction solution to prevent the membrane from drying out.
    Blocking insufficient Increase the blocking incubation time
    Inappropriate blocking buffer Switch different blocking buffer
    Buffer solution has been contaminated Use new buffer solution
    Incomplete washing Increase washing time and washing buffer’s volume
    The antibody concentration may be too high Decrease the concentration of primary antibody or secondary antibody
    Cross-reaction between antibody and blocking agent Choose blocking solution without cross-reaction. Add Tween-20 to the washing buffer to reduce cross reaction.

     

    High background:          Improved:

         

     2.   Low or no signal

     

    Causes solutions
    Insufficient antigen Increase amount of loading samples
    Protein degradation  Re-prepare samples
     The antigen is blocked by blocking buffer  Optimize blocking solution, decrease blocking time or  decrease the concentration of proteins in the blocking solution.
     No or low level of target protein in samples  Run a positive control. If the level of target protein in samples is low, try to increase amount of loading sample.
     Wrong choice of membrane  Choose suitable pore size membrane. Use 0.45um size membrane for proteins larger than 22KD. Use 0.2 µm size membrane for proteins smaller than 22 KD.
     Poor transfer of protein to membrane  Make sure there are no air bubbles between the gel and membrane during transfer. Always ensure assembling electrode correctly. Control transfer temperature and optimize transfer electricity and time.
     Methanol concentration may be too high.  Too high concentration of methanol may result in the separation of protein and SDS and thus cause protein precipitation  in the gel. At the same time, it may cause shrinking or hardening of the gel to inhibit transferring of high molecular weight proteins. As a result, choose suitable methanol concentration according to different molecular weight.
     Excessive washing of membrane  Reduce washing time and washing times
     Over blocking  Lower the concentration of your blocking solution and shorten blocking time. Change blocking solutions.
     The primary antibody is inactive  Use effective antibody in expiration, avoid freezing- thawing repeatedly, and use fresh solution.
     Insufficient reaction of antibody to membrane  Increase the concentration of the antibody and the incubation time.
     The reagents are not compatible with each other  Primary antibody and species, primary antibody and secondary antibody, or enzyme and substrate are not compatible. Setting loading control can validate the secondary detecting system.
     HRP Inhibited  Avoid sodium azide in all solutions and containers
     Enzyme or substrate is inactive.  Directly mix enzyme and substrate. If no color, the enzyme doesn’t work. Choose active conjugated reagent. Use fresh substrate.
     Exposure time is too short  Increase the exposure time 

     

     Low or no signal     Improved

     

    3. Non-specific bands

    Causes Solutions
    Amount of loading samples is too large Decrease amount of loading samples
    Protein degradation Use fresh samples and use protein inhibitor
    Cells were cultured too many passages to result in protein variation Use primary cells or less passaging cells to run a control
    Antibody Concentration is  too high Decrease the concentration of the primary or secondary antibody
    Cross-reaction Choose monoclonal antibody or affinity purified antibody to ensure antibody specificity
    Existence of the different protein  isoforms Some proteins derived from the same gene have different isoforms. The size of every isoform protein is different.
    Non-specific signal caused by the secondary antibody Run a secondary antibody control or choose other secondary antibody
    Substrate is too sensitive Use suitable substrate
    Exposure time is too long Reduce the exposure time

     

    Non-specific bands:   Improved:

     

    4.Wrong band location

    Causes Solutions
    Existence of dimer or polymer Increase a process or intensity of protein denaturation
    Modification of proteins Modification of proteins, such as glycosylation or phosphorylation, can result in an increase of molecular weight of protein.

      

    5.Invisible dots on the bands

    Causes Solutions
    Air bubbles were trapped in the gap of gel and membrane during transferring. Remove bubbles in the gap of gel and membrane when preparing for transferring.

     

    Invisible dots on the bands:   Improved:

     

    6.Incomplete bands 

    Causes  Solutions
    Substrate is not well-distributed during incubation Even the substrate during incubation

     

    Incomplete bands: Improved:

     

    7.Smile effect of the bands

     

    Causes Solutions
    Migration was too fast during electrophoresis Reduce the voltage to slow down the migration
    Migration was too hot Run the gel in the cold room or in ice

     

    Smile effect of the bands:  Improved:

     

    8.White bands on a black blot

    Causes Solutions
    HRP concentration is too high Decrease the secondary antibody concentration

     

    White bands on a black blot:  Improved:

     

    9.Black dots on the blot

     Causes  Solutions
     Air bubbles were trapped against the membrane during transferring or the antibody is not well distributed during incubation  Try to remove bubbles. Keep shaking when incubating the antibody.
     The blocking agent was not well dissolved.  Filter the blocking agent.
     The antibody reacts with the blocking solution.  Choose suitable blocking solution
     There are aggregates in the HRP conjugated secondary antibody.  Filter the secondary antibody agent and remove the aggregates.

     

    Black dots on the blot:      Improved: 

     

    10.Marker lane is black

    Causes Solutions
    The antibody reacts with the MW marker Add a blank lane between the marker and the adjacent sample lane

     

  • 流式细胞技术(FC)

     

    Flow Cytometry (FC) is a new technology for analysis on individual cells by means of flow cytometers. It allows quantitative analysis by measuring multiple parameters simultaneously. Characterized by without damage to the structure and function of cells, organelles or analytical particles in the measurement and speedy, accurate, sensitive and quantifiable process, it has been widely used in cell counting, cell sorting, and analysis of the molecules on cell surface or intracellular. SAB follows strict validation procedure on every batch of antibody to ensure accurate and credible results in FC.

     

     

    Flow cytometry analysis of Human peripheral blood lymphocytes using Mouse IgG2b Isotype Control, FITC Conjugated mAb #28279.

     

    Flow cytometry analysis of Human peripheral blood lymphocytes using Human CD4/CD8/CD3, FITC/PE/PE-Cy5 Conjugated mAb #28249.

     

     Flow cytometry analysis of Human peripheral blood lymphocytes using Human CD3/HLA-DR, FITC/PE Conjugated mAb #28239.

     

    Flow cytometry analysis of Human peripheral blood monocytes using Human CD64, PE Conjugated mAb #28198.

     

     

  • 免疫荧光技术 (IF)

     

    Immunofluorescence (IF) is used to analyze the sub-cellular localization of a protein within a cell. This technique usually uses a labeled antibody to target the fluorescent dye to its antigen. For the emitted light by the fluorescent dye can be detected in fluorescence microscope, this allows visualization of the distribution of a target protein in the sample. SAB follows stringent validation protocol to ensure IF application.   

     
    Immunofluorescence (IF) validation methods:
          Using appropriate cell lines for analysis
          Stimulus treatment
          Inhibitor treatment
     
    Using appropriate cell lines for analysis
    Immunofluorescence analysis of methanol-fixed MCF7 cells using HER2 (Phospho-Tyr1248) Antibody #11079.
     
    Immunofluorescence analysis of methanol-fixed Hela cells using GSK3β (Phospho-Ser9) Antibody #11002.
     
     
     Immunofluorescence analysis of methanol-fixed MEF cells using Histone H3 (Tri-Methyl-Lys27) Antibody #11582.
     
    Stimulus treatment
    Immunofluorescence analysis of methanol-fixed MEF cells untreated or treated with IFN using STAT3 (Phospho-Tyr705) Antibody #11045.

     

    Inhibitor treatment

    Immunofluorescence staining of methanol-fixed MEF cells untreated or treated with LY2904 using FKHR (Phospho-Ser256) Antibody #11115.

     
     

     

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