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: SUMOylation 2/3 Detection Kit White Paper
Kit contents
The SUMOylation 2/3 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 2/3 Affinity Beads IP Control Beads
| BlastR™ Wash Buffer
| Spin Columns Bead Elution Buffer
| Chemiluminescent Reagent A Chemiluminescent Reagent B Anti-SUMO2/3-HRP antibody
|
Example results
There are many applications for these kits, here we describe an interesting example:
Application 1: Investigate significant SUMOylation 2/3 events
Immunoprecipitation using the Signal-Seeker™ SUMOylation 2/3 Detection Kit
Denatured cell lysates were prepared as previously reported1 from HeLa cells ("HS43": Heat Shock treated 42°C for 10 min, and "CT37": untreated) and HeLa siRNA SUMO knockdown ("KDS2"). 1mg of lysate was used for the immunoprecipitation (IP) of SUMOylated 2/3 proteins. Western blots of immunoprecipitated proteins were developed using anti-SUMO-2/3 antibody (Cytoskeleton cat# ASM23) (A) or anti-Ubc9 antibody (B). The level of SUMO-2/3 conjugates in heat shock treated cells is higher than control, and shRNA SUMO-2 knock-down reduced the level of SUMOylated 2/3 modified proteins. Chemical conjugation of SUMO-2/3 antibody (11G2) to the affinity bead matrix prevents heavy and light chain leaching. Unconjugated free SUMO is also captured by the SUMO-2/3 affinity beads.
(B) Unmodified Ubc9 is visible near 18kDa. High molecular-weight band indicates that Ubc9 is SUMOylated by a single SUMO-2/3 protein. Ubc9 has previously been reported to be a target for SUMOylation1,2.
1. Barysch S. et al. 2014. Identification and analysis of endogenous SUMO1 and SUMO2/3 targets in mammalian cells and tissues using monoclonal antibodies. Nat Protoc. 9(4):896-909
2. Becker J. et al. 2013. Detecting endogenous SUMO targets in mammalian cells and tissues. Nature Struc. & Mol. Biol. 20, 525-531.
• 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 2/3 and other PTMs for target proteins.
For more information contact: signalseeker@cytoskeleton.com
Associated Products:
Signal-Seeker™ Phosphotyrosine Detection Kit (Cat. # BK160)
Signal-Seeker™ Ubiquitination Detection Kit (Cat. # BK161)
Signal-Seeker™: BlastR™ Rapid Lysate Prep Kit (Cat. # BLR01)
Signal-Seeker™ SUMOylation 2/3 Affinity Beads (Cat.# ASM24-beads)
Signal-Seeker™: PTMtrue™ SUMOylation 2/3 Antibody (Cat.# ASM23)
Click on the pdf icon below to download the manual
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
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蛋白的类型为PDGF。其有一种二聚体为PDGF-BB。
请教第一点:我看了Elisa的说明书,现在检测PDGF有两种试剂盒,一种是检测PDGF的,要求的试剂样本为组织匀浆;一种是检测PDGF-BB的,要求的待测样本为血液或血浆。
我现在的实验目的是想更精确,检验组织中的PDGF-BB,但是手中的是人体韧带组织样本,非PDGF-BB试剂盒所要求的组织样本为血浆或血清,我不知道是否可以用PDGF-BB的ELISA试剂盒的来做,所以特地请教:)
请教第二点:市面上有RB公司的96T,2500元;另外有Uscnlife的Elisa试剂盒,96T的3680元,都说是原装进口的,我该如何选择呢?为何差价这么多?谢谢指点!
一、免疫组化试剂盒一般包括:
1、特异性的一抗
2、免疫组化检测系统。
3、有的同时还具备显色系统。
二、不同的公司内容有差别,所以购买时一定了解清楚:
1、一抗是做人的还是兔、鼠的组织;
2、检测系统是ABC/SP/非生物素/其他;
3、试剂盒的内容都有什么?有没有显色系统?
4、如果没有,自己根据前面的检测系统需配哪个显色系统。(其他的辅助试剂如PBS缓冲液、抗原修复液等一般另卖或自配)
三、免疫组化抗体即指特异性一抗,是用来标记的指标,免疫组化过程的其他试剂一律另外配备。
另外:做实验时,如遇出结果困难,记得做阳性片对照,敢于怀疑检测系统或显色系统的问题。
用双氧水去除过氧化物酶时,我是自己配的3%双氧水滴到片上而非将片浸入双氧水,这样可否?
用博士德的DAB染色,配制方法为:1mlH20中加入DAB、H2O2、TBS各一滴,我配制的时候发现将DAB加入H20时很容易沉淀,要震荡才能混匀,且将配好的DAB滴到玻片上时,DAB会结为很小块的微颗粒悬浮在液体中但并不染色,是否DAB有问题?
现在打算换中衫的试剂盒,代理商说中衫的试剂盒包括了二抗和DAB,想请教一下该试剂盒的具体内容有些什么?
万分感谢!