Signal-Seeker™ Acetyl-Lysine Enrichment Kit

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Signal-Seeker™ Acetyl-Lysine Enrichment Kit

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The Signal-Seeker™ line of products 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

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 (1mM) and nicotinamide (1mM) for 6 hours. (1) 16.7ml of AAC04 bead slurry (20mg antibody). (2) 50ml of AAC04 bead slurry (60mg antibody). (3) Anti-acetyl lysine rabbit monoclonal mix (Cell Signaling, 1:100 per manufacturer’s instruction). (4) ImmuneChem acetyl lysine affinity bead (40mg antibody). (5) ImmunChem acetyl lysine bead (80mg antibody). (6) Normal mouse IgG control bead (60mg 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 1mM TSA and 1mM nicotinamide for 6 hours, were isolated using BlastR buffer. IP was performed using AAC04 beads (60mg). 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 (60mg) or mIgG control bead (#CIG02, 60mg) in 1mg of tissue extracts. Enriched proteins were separated by SDS-PAGE and analyzed by western blot with AAC03-HRP (1:3000).

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.

Cat#	Size	Price	Qty	Buy
BK163-S	10 assays	£309.66
BK163	30 assays	£582.66

Additional Information

Property	Value or Rating
Manufacturer	Cytoskeleton, Inc
Storage	4°C

Signal-Seeker™ Acetyl-Lysine Enrichment Kit

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Signal-Seeker™ Acetyl-Lysine Enrichment Kit

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