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: Acetyl-Lysine Detection Kit White Paper
Kit contents
The acetyl-lysine 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 HDAC inhibitor Cocktail Precision Red™ Protein Assay Reagent | Acetyl Lysine Affinity Beads IP Control Beads
| BlastR™ Wash Buffer
| Spin Columns Bead Elution Buffer
| Chemiluminescent Reagent A Chemiluminescent Reagent B Anti-Acetyl Lysine-HRP antibody
|
Example results
There are many applications for these kits, here we describe an interesting example:
Application 1: Investigate significant Acetylation events
Immunoprecipitation using the Signal-Seeker™ Acetyl-Lysine Detection Kit compared to cell signaling acetyl lysine antibody and immunechem acetyl lysine affinity beads
Figure 1. Various acetyl-lysine affinity reagents were used to IP acetylated proteins from Cos-7 cells either treated (+) or untreated (-) with TSA (1 μM) and nicotinamide (1 mM) for 6 hours. (1) 16.7 μl of AAC04 bead slurry (20 μg antibody). (2) 50 μl of AAC04 bead slurry (60 μg antibody). (3) Anti-acetyl lysine rabbit monoclonal mix (Cell Signaling, 1:100 per manufacturer’s instruction). (4) ImmuneChem acetyl lysine affinity bead (40 μg antibody). (5) ImmunChem acetyl lysine bead (80 μg antibody). (6) Normal mouse IgG control bead (60 μg antibody). The total profile of enriched acetylated proteins were eluted and analyzed by western blot with an AAC03-HRP antibody (1:3000). AAC04 performed exceptionally well in enriching a broad range of acetylated proteins whereas the other commercial acetyl lysine enrichment reagents primarily enriched the most abundance acetylated proteins (e.g. acetylated tubulin and histones).
Immunoprecipitation of target specific acetylated proteins using the Signal-Seeker™ Acetyl-Lysine Detection Kit
Figure 2A: A431 cells, untreated (-) or treated (+) with 1 μM TSA and 1 mM nicotinamide for 6 hours, were isolated using BlastR buffer. IP was performed using AAC04 beads (60 μg). Total cell lysate (Input) and immunoprecipitated samples were separated by SDS-PAGE and analyzed by western blot with antibodies against EGFR (Millipore, 1:1000), Hsp90 (Abcam, 1:20,000), P53 (Sigma, 1:2000), and RhoDGI(Millipore,1:1000)
Immunoprecipitation of acetylated proteins from liver and heart tissue using the Signal-Seeker™ Acetyl-Lysine Detection Kit
Figure 2B: Mouse tissue extracts (liver and heart) were obtained with BlastR buffer. IP was performed using AAC04 beads (60 μg) or mIgG control bead (#CIG02, 60 μg) in 1mg of tissue extracts. Enriched proteins were separated by SDS-PAGE and analyzed by western blot with AAC03-HRP (1:3000).
• Pharmacological investigation of acetylating and HDAC enzymes involved in regulation of target proteins.
• Investigate acetylation under a variety of different growth factors or drug treatments.
• Examine the interaction of acetylated target proteins with its downstream effectors.
• Examine crosstalk between acetylation 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™ Acetyl-Lysine Affinity Beads (Cat.# AAC04-beads)
Signal-Seeker™: PTMtrue™ Aceetyl lysine Antibody (Cat.# AAC03)
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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采用抗-HBe抗体包被反应板，加入校准品及被测样本，同时加入定量HBeAg中和抗原，经过振荡孵育,洗板后再加入铕标记的抗-HBe，若标本中抗-HBe浓度高，HBeAg将被大量中和，使最后形成的抗-HBe-HBeAg-铕标记抗-HBe复合物减少。增强液（β－NTA）将标记在抗体上的Eu3+解离到溶液中，Eu3+和增强液中的有效成分形成高荧光强度的螯合物，荧光强度和样本中的抗-HBe浓度成反比。
1、直接竞争，标记抗原，与检测样品中的抗原竞争抗体。
2、间接竞争，标记抗体，固相抗原与液相抗原（样品）竞争标记抗体。
3、定义
间接竞争法的模型：包被抗原，用HRP-抗体与样本一起加入。样本中的Ag与Solid-Ag竞争HRP-Ab，固相吸附的HRP-Ab与样本中的Ag浓度成反比。
直接竞争法的模型：包被抗体，用HRP-抗原与样本一起加入。样本中的Ag与HRP-Ag竞争Solid-Ab，固相吸附的HRP-Ag与样本中的Ag浓度成反比。
4、竞争法的理论基础：是限量抗体。只有在限量抗体基础上，两种抗原才会形成竞争关系。
5、、间接竞争法具备较高灵敏度原因。
直接竞争法里，标记抗原与待测抗原均是液相，与抗体的结合机会是一样的，例如有1份标记抗原与1份待测抗原竞争1份抗体，那么有50%的标记抗原能与抗体结合，所以标记抗原的相对结合率为50%。间接法里，固相抗原与抗体的接触面积较小，固相抗原与待测抗原的结合抗体机会是不平等的，接近顺序饱和法，即只有与待测抗原结合剩余的抗体才会与固相抗原结合，同样有1份固相抗原与1份待测抗原竞争1份抗体时，基本上抗体会被待测抗原中和掉，与固相抗原结合的抗体非常少。固相抗原的相对结合率为0%。因此，间接法的抑制曲线斜率会大于竞争法。
因为抑制率越大则斜率越大，从而灵敏度越大。（假设零管变异5%，以两倍SD为灵敏度限，则为90%相对结合率，则间接法可以在较低的待测抗原浓度达到这一相对结合率，因此灵敏度要高。）
在进行系统放大时，间接法一般可以使用酶标二抗。因为二抗可以针对抗体的多个部位，所以存在放大效应，从而能提高间接法的灵敏度。直接法一般难以进行放大，常用的有生物 素化抗原与酶标亲和素，但模式上似乎不存在放大效应。
6、间接法的高灵敏度难以实现的原因：
双抗体 夹心的免放（IRMA）模式刚出现时，也被模型证明灵敏度优于竞争法的放免（RIA），原因也是较大的斜率，但是IRMA的高灵敏度一直到单抗发展后才得以实现。
