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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对于新手来说购买反转录试剂盒是比较理想的，买一个kit看看说明书操作就可以了。推荐两款kit TAKARA和HaiGene，这两款试剂盒都能对RNA样品中的gDNA有效去除，因此对RNA质量的要求不高。TAKARA的RR047A操作方便，反转录温度为37℃，对于大多数试验来讲是满足要求的。HaiGene的D0401操作要多一个步骤，但其反转录温度是55℃（耐高温的反转录酶），提高了反转录温度使得高GC含量、复杂模板、长mRNA的模板都能有效反转录，因此其反转录效率更高，更能够获得样本中基因的真实表达量。如果后续试验是RealTime PCR、ORF克隆、高GC含量、或者你的待研究基因结构复杂程度未知，还是选用耐高温的反转录酶更理想。对于反转录高手来说，直接购买反转录酶、再购买Rnase Inhibitor自己配制反转录体系就可以了。对于样本量大的课题组来讲，相对还是比较经济的。选择反转录酶时仅需要考虑是否需要耐高温的酶，来克服目的基因的复杂结构就可以了。PROMEGA、TAKARA、HaiGene、 TRANSGEN等品牌的反转录酶性价比还都是不错的。Life、NEB的也不错，不过性价比一般，不一一解释了。
如果是用转录出的cDNA做那跟平时就一样了。不需要特别的试剂盒、
逆转录（reverse transcription）是以RNA为模板合成DNA的过程，即RNA指导下的DNA合成。此过程中，核酸合成与转录（DNA到RNA）过程与遗传信息的流动方向（RNA到DNA）相反，故称为逆转录。逆转录过程是RNA病毒的复制形式之一，需逆转录酶的催化。 逆转录过程的揭示是分子生物学研究中的重大发现，是对中心法则的重要修正和补充。人们通过体外模拟该过程，以样本中提取的mRNA为模板，在逆转录酶的作用下，合成出互补的cDNA，构建cDNA文库，并从中筛选特异的目的基因。该方法已成为基因工程技术中最常用的获得目的基因的策略之一。
