Acetyl-Lysine Affinity Beads - AAC04

Acetyl-lysine Affinity Beads (40 Assays)
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AAC04-Beads

NEW Format -  The new, versatile format allows for more precise control when using the acetyl-lysine affinity beads

As part of the Signal-Seeker™ product line, AAC04 acetyl-lysine affinity beads have been optimized to detect endogenous levels of lysine-acetylated proteins. The new, versatile format of the acetyl-lysine affinity beads are provided as AAC02-Beads (Clone 7B5A1) and AAC03-Beads (Clone 10C4B2.1) that have been chemically conjugated to Protein G beads. These beads can be combined in a 1:1 mixture to produce AAC04-Beads, and the new format provides great flexibility and precision when investigating acetyl-lysine PTMs. Validation studies have shown that these beads can immunoprecipitate a wide range of lysine-acetylated proteins with minimal detectable leaching of either heavy or light chains in an IP assay. A comprehensive Signal-Seeker™ acetyl-lysine Detection Kit is also available (BK163) and is recommended for first time users.

Validation Data: Acetyl-Lysine Affinity Beads White Paper

Each lot of affinity-bead is quality controlled to provide high batch to batch consistency, see COA documents.

Validated Applications

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 with the new AAC04-Beads format

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 with the new AAC04-Beads format

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

Enrichment of total acetyl-lysine proteins using AAC04 affinity beads, cell signaling acetyl lysine antibody, and immune chem 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 ml of AAC04 bead slurry (20 μg antibody). (2) 50ml 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).

acetylation_antibodies_image_3

Amount:
Each package contains enough acetyl-lysine affinity beads for 40 reactions.

 

For more information contact: signalseeker@cytoskeleton.com

Associated Products:

Signal-Seeker™ SUMOylation 2/3 Detection Kit (Cat. # BK162)

Signal-Seeker™ Ubiquitination Detection Kit (Cat. # BK161)

Signal-Seeker™ Acetyl-Lysine Detection Kit (Cat. # BK163)

Signal-Seeker™: BlastR™ Rapid Lysate Prep Kit (Cat. # BLR01)

Signal-Seeker™: PTMtrue™ Aceetyl lysine Antibody (Cat.# AAC03)

For product Datasheets and MSDSs please click on the PDF links below.

AuthorTitleJournalYearArticle Link
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
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

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