The Signal-Seeker™ line of produts have been developed to allow simple analysis of key regulatory protein modifications by specialists and non-specialists alike. The comprehensive Signal-Seeker™ kits provide an affinity bead system to isolate and enrich modified proteins from any given cell or tissue lysate. The enriched protein population is then analyzed by standard western blot procedures using a primary antibody to the target protein.
Product Uses Include
- Investigate transient regulatory mechanisms
- Measure signalling events of multiple pathway member proteins
- Discover new modifications of your protein of interest
- Gain insight into regulatory mechanisms
- Measure endogenous or transiently expressed protein signalling events
Validation Data: see datasheet
Kit contents
The SUMOylation 1 kit contains the following components:
| Lysis and protein quantitation step | IP and pre-clear step | Wash step | Elution step | Western step |
BlastR™ Lysis Buffer BlastR™ Dilution Buffer BlastR™ Filters Protease Inhibitor Cocktail De-SUMOylation inhibitor Cocktail Precision Red™ Protein Assay Reagent | SUMOylation 1 Affinity Beads IP Control Beads
| BlastR™ Wash Buffer
| Spin Columns Bead Elution Buffer
| Chemiluminescent Reagent A Chemiluminescent Reagent B Anti-SUMO1 antibody
|
Example results
There are many applications for these kits, here we describe an interesting example:
Application 1: Investigate significant SUMOylation 1 events
Immunoprecipitating total SUMO1 profiles using the Signal-Seeker™ SUMOylation 1 Detection Kit compared to older SUMO1 tools (A) HAP1 wildtype (WT) or SUMO1 knockout (KO) lysate, was obtained using BlastR lysis and filter system. 1 mg of each lysate were incubated with 40 µg of each SUMO1 affinity reagent: ASM11-beads (Cytoskeleton), 21C7 (Invitrogen—purified), 21C7 (DSHB—supernatant), D11-beads (Santa Cruz) and conjugated SUMO1 IgG control beads (CIG03). 21C7 antibodies were captured with protein G agarose beads to enrich for SUMO-1 modified proteins. Samples were separated by SDS-PAGE and transferred to PVDF. Enriched SUMO1 samples were analyzed by western blot using ASM01 (Cytoskeleton) antibody at 1:5000, and mouse Trubelot Ultra-HRP secondary at 1:1000 in 5% milk. Trueblot secondary was used to minimize heavy and light chain detection from 21C7 samples. (B): IP was performed using ASM11 as in Fig 1A. SUMO1 modified proteins were visualized with ASM01 1:5000, and anti-mouse secondary at 1:20,000 to highlight the profile of SUMOylated proteins in the region between 64-30 kDa that may be masked by heavy and light chain interference when using unconjugated antibodies for IP. |
Immunoprecipitating SUMO1 modified target proteins using the Signal-Seeker™ SUMOylation 1 Detection Kit compared to older SUMO1 tools
HAP1 wildtype (WT) or SUMO1 knockout (KO) lysate, was obtained using BlastR lysis and filter system. 1 mg of each lysate were incubated with 40 µg of each SUMO1 affinity reagent: ASM11-beads (Cytoskeleton), 21C7 (DSHB—supernatant), and conjugated SUMO1 IgG control beads (CIG03). 21C7 antibodies were captured with protein G agarose beads to enrich for SUMO-1 modified proteins. Samples were separated by SDS-PAGE and transferred to PVDF. Target proteins: (A) TFII-I, RanGAP1, and (B) schmd1 were analyzed for their SUMO1 modified forms by western blot. Anti-rabbit-HRP labeled secondary antibody was used at 1:10,000. All three primary antibodies are rabbit polyclonal antibodies, and should not bind heavy and light chain fragments from the IP antibody.
• Pharmacological investigation of SUMOylating and de-SUMOylating enzymes involved in regulation of target proteins.
• Investigate SUMOylation under a variety of different growth factors or drug treatments.
• Examine the interaction of SUMOylated target proteins with its downstream effectors.
• Examine crosstalk between SUMOylation 1 and other PTMs for target proteins.
For more information contact: signalseeker@cytoskeleton.com
Associated Products:
Signal-Seeker™ SUMOylation 2/3 Detection Kit (Cat. # BK162)
Signal-Seeker™ Ubiquitination Detection Kit (Cat. # BK161)
Signal-Seeker™: BlastR™ Rapid Lysate Prep Kit (Cat. # BLR01)
Signal-Seeker™ SUMOylation 2/3 Affinity Beads (Cat.# ASM11-beads)
Signal-Seeker™: PTMtrue™ SUMOylation 2/3 Antibody (Cat.# ASM01)
Click on the pdf icon below to download the manual 
New product released in 2018! For the most recent publications citing this and other Signal-Seeker™ products, see our Signal-Seeker™ Validation Data Page click here
Visit our Signal-Seeker™ Tech Tips and FAQs page for technical tips and frequently asked questions regarding this and other Signal-Seeker™ products click here
If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com
ebiomall.com
>
>
>
>
>
>
>
>
>
>
>
>
1.步骤简单，45分钟内完成测定，比经典Lowry法快4倍而且更加方便。
2.灵敏度高，检测浓度下限达到25μg/ml，最小检测蛋白量达到0.5μg，待测样品体积为1-20μl 。
3.BCA法测定蛋白浓度不受绝大部分样品中的去污剂等化学物质的影响，可以兼容样品中高达5%的SDS，5%的Triton X-100，5%的Tween 20，60，80。
4.在20-2000μg/ml浓度范围内有良好的线性关系。
5.检测不同蛋白质分子的变异系数远小于考马斯亮蓝法蛋白定量。
定性ELISA只能粗略表示样本待测物含量在某个特定值以上或以下，定性ELISA通常设置阳性对照（P）、和阴性对照（N），结果分别用“阴性”和“阳性”表示。ELISA定性测定的“阴性”和“阳性”的判断标准是试剂盒所确定的阳性判断值，即cut-off值。
定量ELISA是通过一系列不同已知浓度的标准品所对应的OD值做出标准曲线，然后将样本的OD值代入标准曲线，计算出样本中待测物的含量。百特纯大分子Meretciel的定量ELISA试剂盒还可以。
加入荧光标记探针，巧妙地把核算扩增、杂交、光谱分析和实时检测技术结合在一起，借助于荧光信号来检测PCR产物。一方面提高了灵敏度，另一方面还可以做到PCR每循环一次就收集一个数据，建立实时扩增曲线，准确地确定CT值，从而根据CT值确定起始DNA的拷贝数，做到真正意义上的DNA定量。另外由于CT值是一个完全客观的参数，CT值越小，模版DNA的起始拷贝数越小。因此，利用CT值确定DNA拷贝数实时PCR方法比普通终点定量方法更加准确
临床实验室对商品定量试剂盒分析性能的验证.pdf(17431.6k)
1.模板提取（一般为RNA）：Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定：分光光度计或NanoDrop
3.逆转录：逆转录试剂盒（或者一步法试剂盒），这一步可以用普通PCR做，也可以用水域做。
4.荧光定量PCR试剂：通常有用SYBR Green Mix做的，但是这里建议你用EvaGreen做，灵敏度和平行性都要好于SYBR Green，并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他：除了以上的那些还需要离心管、PCR管或板（Axygen反应比较好）、移液枪等，暂时就想到这么多。
至少可以说同一种蛋白定量试剂盒并不适用于所有细胞
蛋白定量试剂盒有分Bradford，BCA，Lowry等，每一种对不同的细胞都有独特的适用范围和灵敏度。所以需要多种蛋白定量试剂盒对比检测才能让结果准确
