Signal-Seeker™ Acetyl-Lysine Detection Kit (15 assay)
NEW Format - The new, versatile format allows for more precise control when using the acetyl-lysine affinity beads
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
There are many applications for these kits, here we describe an interesting example:
Application 1: Immunoprecipitation of total Acetyl-Lysine Profile Detection Using AAC02-Beads, AAC03-Beads or combining the beads (AAC04-Beads) -- All 3 options are possible when using the BK163 Kit
AAC02-Beads, AAC03-Beads, and AAC04-Beads (50µl bead slurry) were used to IP acetylated proteins from Cos-7 cells either treated (+) or untreated (-) with deacetylase inhibitors [TSA (1µM) and nicotinamide (1mM)] for 6 hours. The total profile of enriched acetylated proteins were eluted and analyzed by western blot with an AAC03-HRP antibody (1:3000). Mouse IgG beads are used as a control for non-specific binding (Cat # CIG02). Each IP assay utilized 1 mg of Cos-7 lysate.
Application 2: Immunoprecipitation of target-specific acetylated proteins Using AAC02-Beads, AAC03-Beads or combining the beads (AAC04-Beads) -- All 3 options are possible when using the BK163 Kit
AAC03-Beads & AAC02-Beads (50µl bead slurry) and a 1:1 mix (25 µl each) AAC04-Beads, were used to IP acetylated proteins from Cos-7 cells either treated (+) or untreated (-) with deacetylase inhibitors, [TSA (1µM) and nicotinamide (1mM)], for 6 hours. Western blot analysis using anti EGFR and anti-RhoGDI antibodies was performed and signals were quantitated using LiCor Empiria software. IP assays were also carried out and signals quantitated for mouse IgG control beads (blots not shown). Each IP used 1 mg of lysate.
Application 3: Immunoprecipitation of acetylated proteins from tissue samples
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).
Signal-Seeker ™ Acetyl-Lysine Affinity Beads out-performs other acetyl-lysine affinity beads
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).
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-AAC03 Acetyl Lysine Affinty Beads-AAC02 IP Control Beads
| BlastR™ Wash Buffer
| Spin Columns Bead Elution Buffer
| Chemiluminescent Reagent A Chemiluminescent Reagent B Anti-Acetyl Lysine-HRP antibody
|
• 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™ Acetyl-lysine Antibody (Cat.# AAC03)
Click on the pdf icon below to download the manual 
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If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com
