Signal-Seeker™ Acetyl-Lysine Detection Kit (30 assay)

Signal Seeker Acetylation Detection Kit (30 assays, immunoprecipitation format)
$0.00

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.

 

aac04_fig1_2

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.

 

 

aac04_fig_2

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).

bk163_Tissue

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).

aac04_fig2_1

Kit contents

The acetyl-lysine kit contains the following components:

Lysis and protein quantitation stepIP and pre-clear stepWash stepElution stepWestern 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

 

 

 

 

Other experiments that could be attempted in this area of research include:

• 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

 

 

AuthorTitleJournalYearArticle Link
Williams D. et. al.Type II Interleukin-4 Receptor Activation in Basal Breast Cancer Cells Promotes Tumor Progression via Metabolic and Epigenetic ModulationIntl Jrnl of Mol Sci2024ISSN 1422--0067
He, Ying et al.PPARγ Acetylation Orchestrates Adipose Plasticity and Metabolic RhythmsAdvanced Science2023ISSN 2198--3844
Oldfield C. et. al.Muscle-specific sirtuin 3 overexpression does not attenuate the pathological effects of high-fat/high-sucrose feeding but does enhance cardiac SERCA2a activityPhysiol Rep.2021ISSN 3440-5591
Kumar, Manish et al.Inhibition of histone acetyltransferase function radiosensitizes CREBBP/EP300 mutants via repression of homologous recombination, potentially targeting a gain of functionNature Communications2021ISSN 2041-1723
Losson H. et. al.The HDAC6 inhibitor 7b induces BCR-ABL ubiquitination and downregulation and synergizes with imatinib to trigger apoptosis in chronic myeloid leukemiaPharmacol Res.2020ISSN 3261-9722
Vega M. et. al.Stimulation of Fibronectin Matrix Assembly by Lysine AcetylationCells2020ISSN 2073--4409
He, Anyuan et al.Acetyl-CoA Derived from Hepatic Peroxisomal β-Oxidation Inhibits Autophagy and Promotes Steatosis via mTORC1 ActivationMolecular Cell2020ISSN 1097-4164
Aon, Miguel A. et al.Untangling Determinants of Enhanced Health and Lifespan through a Multi-omics Approach in MiceCell Metabolism2020ISSN 1932-7420
Horita, Henrick et al.Utilizing a comprehensive immunoprecipitation enrichment system to identify an endogenous post-translational modification profile for target proteinsJournal of Visualized Experiments2018ISSN 1940-087X
Horita, Henrick et al.Utilizing optimized tools to investigate PTM crosstalk: Identifying potential PTM crosstalk of acetylated mitochondrial proteinsProteomes2018ISSN 2227-7382

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