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。

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.

           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。

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。

Pathway description:

Akt (v-Akt Murine Thymoma Viral Oncogene)/ PKB (Protein Kinase-B) is a Serine/threonine Kinase that is involved in mediating various biological responses, by phospho-rylation of a number of intracellular proteins, regulates different cellular processes, such as cell growth, cell cycle, apoptosis and glucose metabolism. Akt is activated by several hormones including insulin, growth factors, by signals derived from receptors for extracellular matrix molecules such as integrins, by several forms of cellular stress such as oxidative stress or cell swelling, and by activation of Ras. Three mammalian isoforms are currently known: Akt1/PKB- Alpha, Akt2/PKB-Beta and Akt3/ PKB-Gamma. All three isoforms of Akt share a common structure of three domains. Activation of PI3K by receptor tyrosine kinases mediates the phosphorylation of PIP2 to PIP3 and thereby the recruitment of Akt/PKB and PDK to the plasma membrane. Akt/PKB is subsequently activated and phosphorylation at Thr308 by PDK. PIP3 at the cell membrane recruits protein kinases such as Akt/PKB and PDK.PTEN functions as an antagonist of PI3K.PDK further activates SGK and aPKC , aPKC and SGK in turn phosphorylate a wide variety of cellular signaling molecules relevant for the regulation of cell growth, cell cycle and cell proliferation, including FKHR, GSK3, mTOR and p70S6K, for apoptosis, including Bad, caspase 9, IκB, FKHR, Mdm2. Akt/PKB further activates mTOR, a kinase stimulating the uptake of nutrients such as glucose, amino acids, cholesterol and iron. mTOR regulates the phosphorylation of p70S6K which can similarly be activated by PDK. mTOR further activates eIF4E-binding protein-1 and thus is involved in the regulation of translation.   

Selected Reviews:

Wang Q,Liu L,et al.(2003)Control of synaptic strength,a novel function of Akt.Neuron.38(6),915.  

Basso Ade, Solit DB,et al.(2002)Akt forms an intracellular complex with heat shock protein 90 (Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function.J Biol Chem.277(2),39858.

Fornaro M,Plescia J,et al.(2003)Fibronectin protects prostate cancer cells from tumor necrosis factor-alpha-induced apoptosis via the AKT/survivin pathway. J Biol Chem.278(50),50402. 

Pommery N,Henichart JP.(2005)Involvement of PI3K/Akt pathway in prostate cancer –potential strategies for developing targeted therapies.Mini Rev Med Chem.5(12),1125

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 μ g/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。检测细胞内成分，用PBS细胞悬液稀释后，使细胞浓度达到约100万/ mL。 通过超声处理，使细胞膜受损并释放细胞内成分，以1000×g离心20分钟，收集上清液。 保存过程中如果形成沉淀，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℃ 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。

背景介绍

蛋白免疫印迹( Western Blot)是将PAGE(聚丙烯酰胺凝胶电泳)分离的蛋白质样品，转移到固相载体(例如硝酸纤维素薄膜)上，固相载体以非共价键形式吸附蛋白质，且能保持电泳分离的多肽类型及其生物学活性不变。以固相载体上的蛋白质或多肽作为抗原，与对应的抗体起免疫反应，再与酶或同位素标记的第二抗体起反应，经过底物显色或放射自显影以检测电泳分离的特异性目的基因表达的蛋白成分。

一． 仪器与材料

1.        仪器

      1) 电泳仪   2)电转仪   3)摇床   4)化学发光成像仪

2.        材料

11)     洗涤液：TBST（1×TBS，0.1%TWEEN20）

12)     封闭液，抗体稀释液：5%脱脂奶粉的TBST

13)     HRP-羊抗兔IgG

14)     ECL底物液A

15)     ECL底物液B

二．  操作步骤

1.        组装制胶平台

玻璃板用自来水清洗干净，晾干。去离子水检漏后，用滤纸吸去玻璃板中残留的水。

2.       配制所需浓度分离胶（配方见附表）

注意：如果发现10%SDS有沉淀，可先放置50℃左右的温箱或温水浴中至沉淀溶解，待其冷却后方能配胶； 胶液配好后要充分混匀。

3.       灌分离胶

灌胶时要防止气泡产生，每次不要把枪头中液体完全打尽，以免带进气泡，一块0.75mm厚度的胶需加入约4ml的分离胶，灌完后在胶面上轻轻加入2ml蒸馏水封胶。室温凝胶30-40分钟。胶凝后将封胶水倒掉，并用滤纸吸干微量剩余的水。

4.       配制 5% 浓缩胶（配方见附表）

5.       灌浓缩胶

将浓缩胶加满至整个制胶平板，插上合适的梳子。室温凝胶30分钟后。将玻璃板取出，并将玻璃板反转置于内侧。在电泳缓冲液中将梳子拔出。

6.       样品准备

同时进行样品处理 (样品与5×上样缓冲液按照体积比2: 1混合)，100℃煮沸5分钟。将处理好的样品放置4℃冰箱预冷5分钟。如有沉淀，则将样品离心弃沉淀。

7.       上样

在电泳芯内层玻板之间灌满电泳缓冲液；依次点上样品及Marker。

8.       电泳：90V恒压跑浓缩胶，然后120 V恒压跑分离胶

9.       转膜准备

剪好大小与胶相同的1张NC膜和4层滤纸，将膜取出（要用镊子，不可以用手直接接触膜表面）先在水中浸泡10分钟，以除去气泡；然后置于1×转移缓冲液中平衡20分钟。再将滤纸于1×转移缓冲液中浸透平衡，时间不宜过长。按照滤纸-凝胶-膜-滤纸三明治式放好。胶和膜之间不能有气泡。膜在正极，胶在负极。

10.    转膜

将预冷的转移缓冲液加入到转印槽中，按所需条件调好仪器参数，在放置了冰块的水中进行转膜。

11.    收膜和封闭

转膜终止，取出NC膜并用TBST清洗，做好标记后可根据需要置于立春红染液中观察转膜效果，观察完用TBST清洗，然后放入加有封闭液的容器中，室温封闭1 hr左右。封闭后用TBST清洗，或晾干备用保存至-20℃冰箱。

12.    切膜

将转好的膜用TBST先浸泡透，取出后将转有蛋白的一面朝上放置在平板上，用刀按方案所需将膜裁切，并在无蛋白位置作好标记（包括抗体编号，膜编号等），分别放入到各反应槽中。

(磷酸酶实验CIP)

用水稀释10x 磷酸酶buffer至1x工作液，以每个泳道50单位CIP配5ml工作液的体系孵膜，37℃孵

育2小时。将孵完CIP的膜在TBST中洗净。

13.    孵育一抗

将各个反应槽中加入所需反应体积的5%脱脂奶粉TBST，然后按照设计方案的稀释度分别加入相应体积的一抗样品。4℃缓慢摇晃过夜。

14.    洗涤

在各个反应槽中加入适量的TBST，摇床摇动5分钟，然后将TBST倒掉，重复3次。

15.    孵育二抗

配制二抗孵育液：将HRP-羊抗兔IgG二抗按照工作稀释比例加到5%脱脂奶粉TBST中，摇床混匀5分钟。将洗涤过的NC膜全部取出，转移到二抗孵育液中室温1小时。

16.    洗涤

倒掉二抗孵育液，加入适量TBST，摇床摇动5分钟，然后将TBST倒掉，重复3次。

17.    底物反应

配制ECL反应底物：临用前，将ECL底物液A 和ECL底物液B等体积混合并混匀，避光保存备用。

将配好的反应底物液均匀的滴加在膜上，保证所有膜都被反应液完全覆盖后，反应1-2分钟，注意膜的位置不要有气泡。

18.    曝光

预先打开凝胶成像仪，调节至最佳条件，将放有膜的平板放入暗箱内，开始曝光。根据情况选择最适的曝光时间。

19.    结果保存

选择最佳图片，整理归档。

Deparaffinization

1) Incubate slide at 60 degree for 60 minutes.
2) Deparaffinize in Xylene for 10 minutes and repeat one more times.
3) Hydrate in 100% alcohol for 5 minutes, in 95% alcohol for 5 minutes, in 85% alcohol for 5 minutes, in 75%
alcohol for 5 minutes.
4) Dip into Distill Water for 5 minutes.
5) Dip into TBS (50 mM Tris, 100 mM NaCl, pH 7.6), leave for 5 minutes, and repeat two times.

Antigen Retrieval

6) Bring 500 - 2000 ml 10 mM citrate buffer (pH6.0) to the boil in a stainless steel pressure cooker.
7) Put the slide into staining rack and lower into pressure cooker ensuring the slide is well immersed in citrate
buffer.
8) When the pressure indicator valve has risen after 3-4 minutes, incubate for 1 minute.
9) Cool the slide naturally to room temperature.
10) Dip into distilled water, leave for 5 minutes, and repeat two times.
11) Dip the slide in TBS for 5 minutes and repeat two times.
12) Immerse slides in 3% H2O2 ( in fresh methanol) for 15 minutes at room temperature.
13) Wash with distilled water two times, 5 minutes each time.
14) Wash with TBS (pH 7.6) two times, 5 minutes each time.

Staining with Primary Antibody

15) Place the slide into Blocking Solution (3% BSA in TBS) for 30 minutes.
16) Dilute primary antibody with 3% BSA in TBS. Cover the tissue section on the slide with diluted primary
antibody (use 50 – 150 μl for each slide).
17) Incubate at 37degree for 30 minutes or at room temperature for 60 minutes (The optimal incubation time,
incubation temperature, and antibody dilution should be determined by the individual laboratory).
18) Wash with TBS two times, 5 minutes each time.

Staining with Secondary Antibody

19) Incubate with 100-200μl biotinylated secondary antibody diluted in Blocking Solution. Incubate 30 minutes at
37degree.
20) Wash with TBS for 3 times, 5 minutes each time.
21) Incubate with 100-200μl streptavidin- HRP and incubate 30 minutes at 37degree.
22) Wash with TBS for 3 times, 5 minutes each time.
23) Add DAB solution and incubate 10 minutes(The reaction progress and the optimal time should be determine
according to microscope);
24) Wash with distilled water for 2 times, 5 minutes each time.
25) Counterstain sections in hematoxylin if required,wash with distilled water.Immerse slides in 0.1% HCl-
ethanol for 1-10 seconds, wash with distilled water .
26) Dehydrate through 95% ethanol for 1 minute, 100% ethanol for 2×3min, Xylene for 2×3min, and coverslip
with mounting medium.

操作步骤

(推荐#CK0062 免疫组化试剂盒)，CK0063免疫组化笔

1、组织固定：在脱蜡之前，将切片 60°C 恒温箱中烘烤 60 分钟。

2、脱蜡：将切片用二甲苯浸泡 10 分钟，更换二甲苯后再浸泡 10 分钟。

3、水化：分别用无水乙醇浸泡 5 分钟；95%乙醇中浸泡 5 分钟；85%乙醇中浸泡5 分钟；75%乙醇中浸泡5分钟。

4、洗涤：ddH2O 浸泡 5 分钟，清洗 3 次。

5、抗原修复（煮沸法）：压力锅中加入足以淹没切片的 10 mmol/L 的枸橼酸盐缓冲液（pH 6.0）（配制：将复性剂溶于相应体积 ddH2O 中，混匀），加热至沸腾，切片置耐热塑料切片架上，放入锅中，盖好锅盖，扣上压力阀，继续加热，设置保压 3 分钟，时间到后打开放气阀放气，压力归零后开锅盖将内锅取出放置室温冷却，待溶液冷却至室温后取出切片（约 30 分钟）。

6、洗涤：ddH2O 浸泡 5 分钟，清洗 2 次，PBST 浸泡 5 分钟，清洗 2 次。 www.sabbiotech.com 成份 20 片 50 片复性剂（溶解前请混匀） 6 g （溶于 2 L ddH2O） 15 g（溶于 5 L ddH2O）HRP 标记的二抗（抗兔/小鼠） 500 μl 1.25 ml DAB 缓冲液（20X） 50 μl 125 μl DAB 底物（20X） 50 μl 125 μl DAB 色原（20X） 50 μl 125 μl 苏木素体细胞快速染色液 1 ml 2.5 ml For Research Use Only Order:order@signalwayantibody.com Support:tech@signalwayantibody.com

7、灭活酶：每张切片滴加 50 μl 灭活酶试剂，室温避光处理 15 分钟。

8、洗涤：PBST 浸泡 5 分钟，清洗 3 次。（第一次迅速倒掉）

9、封闭：每张切片滴加 3% BSA-PBST 50 μl，湿盒中室温封闭 30 分钟。

10、加一抗：弃去封闭液，每张切片滴加 50 μl 一抗，4°C 湿盒中孵育过夜或 37°C 1 小时。

11、复温：次日取出切片，室温复温 40 分钟。（复温仅针对一抗 4°C 孵育过夜，37℃孵育直接进行下一步）

12、洗涤：PBST 浸泡 5 分钟，清洗 3 次。（第一次迅速倒掉）

13、加酶标二抗：每张切片滴加 HRP 标记的二抗（抗兔/小鼠）25 μl，室温或37°C 孵育30 分钟。

14、洗涤：PBST 浸泡 5 分钟，清洗 3 次。（第一次迅速倒掉）

15、显色（DAB 法）：每张切片滴加 50 μl 显色液（显色液配制：85% ddH2O+ 5% DAB 缓冲液+5%DAB底物+5%DAB色原，避 光现配现用，按需要量配制），湿盒中显色 5 分钟。

16、终止显色：用蒸馏水终止显色反应。

17、复染：每张切片滴加 50 μl 苏木素体细胞快速染色液，染色 5 分钟，用蒸馏水冲洗干净。

18、脱色反蓝：将切片放入 1%盐酸~乙醇中脱色 3~5 秒后迅速取出放入蒸馏水中终止，再放入PBST 中反蓝5分钟。

19、封片：分别在 75%乙醇中浸泡 5 分钟；85%乙醇中浸泡 5 分钟；95%乙醇中浸泡5 分钟；无水乙醇中浸泡5分钟。用二甲苯浸泡 10 分钟，更换二甲苯后再浸泡 10 分钟。之后在切片上加中性树胶，加盖玻片。

20、观察：显微镜。

注意事项

 抗原修复后需冷却，避免洗涤时脱片。

 加抗体时避免交叉污染，导致假阳性。

 显色时间可根据实际染色效果来确定，不一定遵循规定的时间。

 封片时不要产生气泡，以免影响观察。

 显微镜观察时可在低倍镜下先找好视野，再换高倍镜拍照。

 如应用于冰冻切片，请用 4°C 丙酮固定 10 分钟，然后从第 6 步开始操作。

De-Paraffinization and Antigen Retrieval
1.    Incubate slide at 60°C for 60 min.
2.    Bath the slides in antigen retrieval solution and heat it to boil with a microwave on medium power.
       Once boiling, immediately transfer to a small power and keep faint boiling while heating for 2-3 min.
3.    Cool them to room temperature and wash rocking in distilled water several times.
4.    Immerse slides in 3% H2O2 (in fresh methanol) for 15 min at room temperature.
5.    Wash with TBS (pH 7.6) three times, 5 min each time.
 
Staining with Primary Antibody
1.    Dilute primary antibody with 3% BSA in TBS. Cover the tissue section on the slide with diluted primary
       antibody (use 50 – 150μl for each slide).
2.    Incubate at 37°Cfor 2 hours (The optimal incubation time, incubation temperature, and antibody dilution
       should be determined by the individual laboratory).
3.    Wash with TBS three times, 5 min each time.
 
Staining with Secondary Antibody
1.    Incubate with 100-200μl Polymer Enhancer. Incubate 30 min at room temperature.
2.    Wash with TBS for 3 times, 5 min each time.
3.    Incubate with 100-200μl Polymerized HRP and incubate 30 min at 37°C.
4.    Wash with TBS for 3 times, 5 min each time.
5.    Add DAB solution and incubate 3-5 min(The reaction progress and the optimal time should be determine
       according to microscope).
6.    Wash with distilled water for 2 times, 5 min each time.
7.    Counterstain sections in hematoxylin if required,wash with distilled water.Immerse slides in 0.1% HCl-
       ethanol for 1-10 seconds, wash with distilled water. Doing a counterstain of sections in hematoxylin for
       1-2 min and wash them with distilled water.
8.    Dehydrate through 100% ethanol for 5 min, 90% ethanol for 5 min, 75% ethanol for 5 min, and coverslip
       with mounting medium.

 

Immunofluorescence Solutions and Reagents

A.  MOWIOL (anti-fade agent)  Calbiochem # 475904
1.    Place 6 grams glycerol in a 50 mL tube containing a small stirring bar.
2.    Add 2.4 grams Mowiol and stir to mix.
3.    While stirring add 6 mL ultrapure water and leave 2 hours at room temp.
4.    Add 12 mL 0.2 M Tris, pH 8.5 and 230 mL 1 %Thimerosal (w/v in water).
5.    Incubate in hot water (50 °C) for 10 min. with frequent stirring.
6.    Centrifuge at 5000 g for 15 min. to clarify stirring to dissolve the Mowiol. This can be repeated over
       several hours to get most of the Mowiol into solution. Store as 2 mL aliquots in glass tubes at -20 °C.
       These are stable for about 1 year.
 
 Warm tube to room temperature before use. After use, store at 4 °C for about 1 month. Discard once crystalline deposits are seen in slides or tubes.

Immunofluorescence Protocol

1.    Plate adequate number of cells into 8-well Lab-Tek. II - Chamber Slide. System least 24 hours before
       fixation.
2.    Aspirate medium from wells.
3.    Wash with 1XPBS pH7.4 for two times.
4.    Fix the cells by adding 250ul pre-cold methanol in each well in -20 °C and incubate for 20 min at -20 °C.
5.    Wash the wells once with 0.5 mL PBS, then aspirate. Add a second 0.5 mL PBS to each well and let
        incubate 5 min.
6.    Add 200ul 1% BSA in PBS to each well and incubate 1.5 hours at room temperature.
7.    Wash the cells twice with PBS and aspirate medium.
8.    Add 150ul primary Ab (in PBS + 1% BSA) to each well and incubate for 2 hours at 37°C or 4 °C
       overnight.
9.    Wash the cells with 0.5 mL PBS with gentle shaking for 5 min. at room temperature (do this 3 times).
 
The Following steps should be performed with as little exposure to light as possible

10.    ncubate the cells with 200ul appropriate secondary Ab conjugated to fluorophore Alexa594 or Alexa
         488 from Invitrogen (in PBS + 1% BSA) and incubate for 20 min at room temperature.
11.    Dyes such as Hoechst (diluted from stock to stain the nucleus  (10 mg/mL Hoechst stock solution) of
         this incubation to make the final concentration 1.5ug/ml.
12.    Wash the cells 3 times with 0.5 mL PBS with gentle shaking for 5 min.
13.    Aspirate as much PBS out of the wells as possible.
14.    Place a drop of mounting medium (Mowiol solution brought to room temperature) on to the middle of the
         Chamber Slide. System.
15.    Put appropriate glass coverslips on the top the plate.
16.    Let slides dry at room temperature (overnight in dark) and examine the cells under a fluorescence
         microscope.
 

Solutions and Reagents

1.    PBS: Phosphate Buffered Saline

2.    Incubation Buffer: PBS with 0.5%BSA

3.    Wash buffer: PBS with 1% BSA(or 1% FBS)

4.    Cell fixation: 2% formaldehyde solution

5.    RBC lysis buffer: Red Blood Cell Lysis Buffer

Flow Cytometry(FC) Protocol For Peripheral Blood Sample

1.    Aliquot 1 x 10⁶ cells by volume in a assay tube per test from peripheral blood sample which was
       anticoagulated by EDTA.

2.    Add 2–3ml wash buffer to each tube and rinse by centrifugation. Repeat.

3.    Resuspend cells in 100 µl incubation buffer in a 5ml assay tube per test.

4.    Add fluorochrome, or biotinylated-conjugated primary antibody at the appropriate dilution (see the
       antibody datasheet for the appropriate dilution, such as 10 μl/Test) to the assay tubes.

5.    After mixing, incubate for 20 minutes at room temperature away from light.

6.    Add 2ml 1×RBC lysis buffer to the tube, after mixing incubate it for 10 minutes away from light for
       dissolution of red blood cells.

7.    Discard the supernatant by centrifugation and repeat wash by centrifugation in 2–3 ml  wash buffer buffer.

8.    Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

Flow Cytometrys(FC) Protocol For Cells Sample

1.   Aliquot 1 x 10⁶ cells from pretreated sample in 100ul by volume in a 5ml assay tube per test.

2.   Add fluorochrome, or biotinylated-conjugated primary antibody at the appropriate dilution (see the
      antibody datasheet for the appropriate dilution, such as 10 μl/Test) to the assay tubes.

3.   Wash by centrifugation in 2–3 ml wash buffer.

4.   Resuspend cells in 0.3-0.5 ml PBS and analyze on flow cytometer.

Notices:

Since applications vary, results can be optimized through titrating the appropriate dilution by analyzer.

 背景介绍

蛋白免疫印迹( Western Blot)是将PAGE(聚丙烯酰胺凝胶电泳)分离的蛋白质样品，转移到固相载体(例如硝酸纤维素薄膜)上，固相载体以非共价键形式吸附蛋白质，且能保持电泳分离的多肽类型及其生物学活性不变。以固相载体上的蛋白质或多肽作为抗原，与对应的抗体起免疫反应，再与酶或同位素标记的第二抗体起反应，经过底物显色或放射自显影以检测电泳分离的特异性目的基因表达的蛋白成分。

一． 仪器与材料

1.        仪器

      1) 电泳仪   2)电转仪   3)摇床   4)化学发光成像仪

2.        材料

11)     洗涤液：TBST（1×TBS，0.1%TWEEN20）

12)     封闭液，抗体稀释液：5%脱脂奶粉的TBST

13)     HRP-羊抗兔IgG

14)     ECL底物液A

15)     ECL底物液B

二．  操作步骤

1.        组装制胶平台

玻璃板用自来水清洗干净，晾干。去离子水检漏后，用滤纸吸去玻璃板中残留的水。

2.       配制所需浓度分离胶（配方见附表）

注意：如果发现10%SDS有沉淀，可先放置50℃左右的温箱或温水浴中至沉淀溶解，待其冷却后方能配胶； 胶液配好后要充分混匀。

3.       灌分离胶

灌胶时要防止气泡产生，每次不要把枪头中液体完全打尽，以免带进气泡，一块0.75mm厚度的胶需加入约4ml的分离胶，灌完后在胶面上轻轻加入2ml蒸馏水封胶。室温凝胶30-40分钟。胶凝后将封胶水倒掉，并用滤纸吸干微量剩余的水。

4.       配制 5% 浓缩胶（配方见附表）

5.       灌浓缩胶

将浓缩胶加满至整个制胶平板，插上合适的梳子。室温凝胶30分钟后。将玻璃板取出，并将玻璃板反转置于内侧。在电泳缓冲液中将梳子拔出。

6.       样品准备

同时进行样品处理 (样品与5×上样缓冲液按照体积比2: 1混合)，100℃煮沸5分钟。将处理好的样品放置4℃冰箱预冷5分钟。如有沉淀，则将样品离心弃沉淀。

7.       上样

在电泳芯内层玻板之间灌满电泳缓冲液；依次点上样品及Marker。

8.       电泳：90V恒压跑浓缩胶，然后120 V恒压跑分离胶

9.       转膜准备

剪好大小与胶相同的1张NC膜和4层滤纸，将膜取出（要用镊子，不可以用手直接接触膜表面）先在水中浸泡10分钟，以除去气泡；然后置于1×转移缓冲液中平衡20分钟。再将滤纸于1×转移缓冲液中浸透平衡，时间不宜过长。按照滤纸-凝胶-膜-滤纸三明治式放好。胶和膜之间不能有气泡。膜在正极，胶在负极。

10.    转膜

将预冷的转移缓冲液加入到转印槽中，按所需条件调好仪器参数，在放置了冰块的水中进行转膜。

11.    收膜和封闭

转膜终止，取出NC膜并用TBST清洗，做好标记后可根据需要置于立春红染液中观察转膜效果，观察完用TBST清洗，然后放入加有封闭液的容器中，室温封闭1 hr左右。封闭后用TBST清洗，或晾干备用保存至-20℃冰箱。

12.    切膜

将转好的膜用TBST先浸泡透，取出后将转有蛋白的一面朝上放置在平板上，用刀按方案所需将膜裁切，并在无蛋白位置作好标记（包括抗体编号，膜编号等），分别放入到各反应槽中。

(磷酸酶实验CIP)

用水稀释10x 磷酸酶buffer至1x工作液，以每个泳道50单位CIP配5ml工作液的体系孵膜，37℃孵

育2小时。将孵完CIP的膜在TBST中洗净。

13.    孵育一抗

将各个反应槽中加入所需反应体积的5%脱脂奶粉TBST，然后按照设计方案的稀释度分别加入相应体积的一抗样品。4℃缓慢摇晃过夜。

14.    洗涤

在各个反应槽中加入适量的TBST，摇床摇动5分钟，然后将TBST倒掉，重复3次。

15.    孵育二抗

配制二抗孵育液：将HRP-羊抗兔IgG二抗按照工作稀释比例加到5%脱脂奶粉TBST中，摇床混匀5分钟。将洗涤过的NC膜全部取出，转移到二抗孵育液中室温1小时。

16.    洗涤

倒掉二抗孵育液，加入适量TBST，摇床摇动5分钟，然后将TBST倒掉，重复3次。

17.    底物反应

配制ECL反应底物：临用前，将ECL底物液A 和ECL底物液B等体积混合并混匀，避光保存备用。

将配好的反应底物液均匀的滴加在膜上，保证所有膜都被反应液完全覆盖后，反应1-2分钟，注意膜的位置不要有气泡。

18.    曝光

预先打开凝胶成像仪，调节至最佳条件，将放有膜的平板放入暗箱内，开始曝光。根据情况选择最适的曝光时间。

19.    结果保存

选择最佳图片，整理归档。

 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-HRP／AP）

洗涤

底物显色或曝光检测

结果分析

1．高背景

2．信号弱或无信号

 3．非特异性条带

4.   条带位置不对

 5.   条带内出现不显影圆点

 6.   条带不完整

 7.   微笑条带

 8.   反影（白色条带，黑色背景）

 9.   背景有黑色斑点

 10.Markera变黑

WB实验的主要试剂

1．WB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

 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： 

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

No staining:	Improved:

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

3.Weak staining 

Weak staining:	Improved:

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

4.Strong Staining

Strong Staining:	Improved:

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

1.High Background

High background:	Improved:

 2.   Low or no signal

Low or no signal	Improved

3. Non-specific bands

Non-specific bands:	Improved:

4.Wrong band location

5.Invisible dots on the bands

Invisible dots on the bands:	Improved:

6.Incomplete bands 

Incomplete bands:	Improved:

7.Smile effect of the bands

Smile effect of the bands:	Improved:

8.White bands on a black blot

White bands on a black blot:	Improved:

9.Black dots on the blot

Black dots on the blot:	Improved:

10.Marker lane is black

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.

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 analysis of methanol-fixed MCF7 cells using HER2 (Phospho-Tyr1248) Antibody #11079.

 

 

Immunohistochemistry (IHC) is commonly used for in situ qualitative or quantitative analysis of a target protein in cells of a tissue section. Thought a labeled antibody that is specific to its antigen and sensitive to color-developing agent, this method is also widely used for analyzing the sub-cellular localization of a protein. SAB offers IHC applicable antibodies which have generally been validated by different tissues and various approaches. This will distinctly save your time in experiment and can avoid waste of samples.

Validation Approaches Include:

      Validation by different tissues

      Blocking peptide control

     

Human breast carcinoma tissue

Immunohistochemical analysis of paraffin-embedded human tonsil carcinoma tissue using VASP (Phospho-Ser157) Antibody #11214

 

Rat hippocampal region tissue