Acetylation is one of the most important post-translational modifications (PTMs) of proteins and is widely present in various intracellular proteins. Acetylation modification involves the addition of an acetyl group to the lysine residues of proteins by acetyltransferases, regulating protein activity, stability, interactions, and subcellular localization. It plays a crucial role in numerous biological processes, such as cell cycle, gene expression, DNA repair, and signal transduction. With increasing research, abnormal acetylation has been associated with many diseases, including cancer, neurodegenerative disorders, and cardiovascular diseases.
Since acetylation modifications are typically of low abundance and the modification sites are distributed in a complex manner, traditional methods often face challenges such as high background signal, poor selectivity, and low enrichment efficiency, leading to poor data accuracy and reproducibility in the research process. The acetyl-lysine enrichment kit utilizes high-affinity acetylation-binding antibodies to efficiently and specifically enrich acetylated proteins, providing more stable and high-quality samples. It is suitable for applications such as acetylation site identification, proteomics analysis, and studies of cell signaling pathways.
Product Overview
The acetyl-lysine enrichment kit by MtoZ Biolabs utilizes specific affinity capture technology to selectively enrich lysine acetylation residues (Ac-K), making it suitable for acetylated proteins in cells, tissue samples, and other biological specimens. The kit is based on acetylation-specific antibodies and achieves enrichment of acetylated peptides through highly selective binding, making it ideal for downstream applications such as mass spectrometry analysis.
Product Details
|
Product Details |
Size |
Storage Conditions |
|
Wash buffer 1 |
30 mL×1 |
4℃ |
|
Wash buffer 2 |
30 mL×1 |
4℃ |
|
Wash buffer 3 |
30 mL×1 |
4℃ |
|
Elution buffer |
3 mL×1 |
4℃ |
|
Antibody |
50 µL×1 |
-20℃ |
|
Beads |
2 mL×1 |
4℃ |
Protocol
The acetyl-lysine enrichment kit adopts a specific affinity capture technology and the entire experimental workflow is simple and time-efficient, making it suitable for different laboratory environments and experimental scales. The following is the recommended standardized procedure.
Peptide-Level Acetylation Enrichment Workflow
1. Sample Preparation
(1) Desalt the enzymatically digested protein sample using a C18 column or equivalent method, then lyophilize.
(2) It is recommended to start with >2 mg of digested protein, and reconstitute in Wash Buffer 2 to ensure complete dissolution of peptides.
2. Binding of Beads and Antibody
(1) Wash the bead material twice with Wash Buffer 1;
(2) Resuspend the beads in 200 µL of Wash Buffer 1, add the appropriate amount of antibody, and incubate at 4°C with rotation for 2 hours;
(3) Centrifuge to pellet the beads and retain the supernatant.
Note: It is recommended to perform a BCA assay on the post-incubation supernatant to confirm complete antibody binding.
3. Peptide Incubation
(1) Add the reconstituted peptides to the bead pellet and incubate at 4°C with rotation for 2 hours.
4. Impurity Washing
(1) Centrifuge to separate the beads;
Note: Users may decide whether to retain the peptide solution depleted of acetylated peptides according to their needs.
(2) Wash the beads twice with Wash Buffer 2 and retain the beads;
(3) Wash again twice with Wash Buffer 3 and retain the beads.
5. Elution of Acetylated Peptides
(1) Transfer the beads to a new collection tube, add 100 µL of Elution Buffer and incubate at room temperature for 10 minutes, then centrifuge to collect the eluate. Add another 100 µL of Elution Buffer 2 and repeat the process to obtain a total volume of 200 µL.
(2) Desalt the enriched acetylated peptide sample using a C18 column or equivalent method, then lyophilize.
6. Downstream Analysis
Resuspend the dried product in 20 µL of 0.1% formic acid and proceed with LC-MS/MS analysis or peptide concentration measurement directly.
Product Notes
(1) The entire procedure should be performed at low temperatures (e.g., on ice) to maintain the stability of acetylation modifications.
(2) All buffers should be pre-chilled and freshly prepared.
(3) Ensure that the beads are thoroughly pre-washed before use.
Features and Benefits
1. High Specificity and Efficiency
Utilizes high-affinity acetylation-specific antibodies to ensure selective recognition and enrichment of acetylated proteins. Capable of efficiently capturing both mono- and poly-acetylated modifications while minimizing non-specific binding, thereby improving enrichment efficiency.
2. User-Friendly Operation
The enrichment workflow is optimized for simplicity and speed, typically allowing completion within a single day. Each step is accompanied by detailed instructions and optimizations, making it well-suited for routine laboratory use.
3. High Recovery Rate
The optimized affinity enrichment protocol and buffer system ensure effective capture of acetylated peptides while maximizing sample recovery.
4. Strong Sample Compatibility
The acetyl-lysine enrichment kit is compatible with various sample types, including cells and tissues, and supports a wide range of species such as humans, mice, and rats, offering broad application potential.
5. High Reproducibility and Consistency
The affinity materials provide consistent performance across batches, and the standardized operation process ensures high reproducibility, making it ideal for long-term and large-scale research projects.
Applications
1. Acetylation Proteomics
Used in large-scale proteomics studies to help researchers identify acetylated proteins and build protein modification databases.
2. Cell Signaling Research
Acetylation plays a role in cellular signal transduction. The acetyl-lysine enrichment kit enables enrichment of acetylated peptides, aiding in the investigation of regulatory mechanisms in intracellular signaling pathways.
3. DNA Repair
In DNA repair studies, the acetyl-lysine enrichment kit is used to enrich acetylated proteins involved in the repair process, facilitating research on the role of acetylation in DNA damage repair.
4. Drug Development
By analyzing the dynamic changes of acetylated proteins, it is helpful for research on acetyltransferases or acetylated protein substrates, providing insights for new drug development.
FAQs
Q1: What Types of Samples Are Compatible with the Kit?
A1: The acetyl-lysine enrichment kit is suitable for a variety of sample sources, including cells and tissues, and can be widely applied in various studies of protein modifications.
Q2: Why Is It Necessary to Use Deacetylase Inhibitors?
A2: Deacetylases may rapidly remove acetyl groups from proteins during sample processing, resulting in the loss of acetylation modifications. The use of deacetylase inhibitors such as TSA or nicotinamide effectively prevents this loss and preserves the acetylation state in the sample.
Q3: Is This Kit Compatible with Multiple Species?
A3: Yes, the kit has good cross-species compatibility and is suitable for samples from mouse, human, rat, and other species without the need for special optimization.
Q4: How Should High- or Low-Concentration Samples Be Handled?
A4: For high-concentration samples, dilution is recommended to avoid oversaturation, which may impair enrichment efficiency. For low-concentration samples, ultrafiltration or vacuum centrifugation can be used to increase protein concentration and optimize the enrichment process.
