Lactylation is a newly identified type of post-translational modification (PTM) that involves the covalent attachment of lactyl groups to proteins. It plays critical roles in regulating cellular metabolism, immune responses, inflammatory processes, and tumor progression. With the advancement of metabolomics and epigenetics, the regulatory functions of lactylation in various pathological conditions have gained increasing attention, making it an emerging focus in proteomics research.
Due to the low abundance and weak specificity of lactylation modifications in cells, traditional enrichment methods often suffer from poor selectivity, high background noise, and low reproducibility. The lactate-lysine enrichment kit utilizes high-affinity lactylation-specific antibodies capable of recognizing specific structural domains. Combined with an optimized buffer system and standardized workflow, the kit enables high-specificity, high-purity, and high-recovery enrichment of lactylated peptides, facilitating further investigation into the biological functions of lactylation within cells.
Product Overview
The lactate-lysine enrichment kit from MtoZ Biolabs utilizes specially designed lactylation-binding materials to specifically enrich peptides containing lactylation modifications, which combines affinity capture technology with efficient washing protocols to effectively eliminate non-specific binding and background noise. With a standardized operating procedure, users can complete the enrichment of lactylated proteins in a short time, providing high-quality samples for subsequent experiments such as mass spectrometry analysis, Western blot, and immunoprecipitation.
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 lactate-lysine enrichment kit features specially designed lactylation-binding materials, offering a simplified experimental workflow with short processing times. It is suitable for different laboratory environments and scales, with the following recommended standardized steps for peptide-level lactylation enrichment:
Peptide-Level Lactylation Enrichment Process
1. Sample Preparation
(1) Desalt the protein samples after enzymatic digestion using a C18 column or an equivalent method, and freeze-dry the samples.
(2) The recommended starting amount is >2 mg of protein digestion products, resuspended in Wash Buffer 2, ensuring that the peptides are fully dissolved.
2. Beads and Antibody Binding
(1) First, clean the beads using Wash Buffer 1, repeating this step twice.
(2) Resuspend the beads in 200 µL of Wash Buffer 1 and add the appropriate amount of antibody. Incubate with rotation at 4°C for 2 hours.
(3) Centrifuge and separate the beads, keeping the supernatant.
Note: It is recommended to quantify the supernatant after incubation using the BCA method to ensure full antibody binding.
3. Peptide Incubation
(1) Add the resuspended peptide to beads precipitation, and incubate at 4 °C for 2 hours.
4. Washing Impurities
(1) Centrifuge and separate the beads.
Note: Users can decide whether to keep the supernatant containing the de-lactylated peptides based on their needs.
(2) Wash the beads twice with Wash Buffer 2, keeping the beads.
(3) Wash the beads twice with Wash Buffer 3, keeping the beads.
5. Elution of Lactylated Peptides
(1) Replace with a new collection tube, add 100 µL Elution Buffer, incubate at room temperature for 10 minutes, centrifuge and collect, then add 100 µL Elution Buffer 2, repeat once, for a total of 200 µL.
(2) Desalt the enriched lactylated peptide samples using a C18 column or an equivalent method, then freeze-dry the samples.
6. Downstream Analysis
Resuspend the dried product in 20 µL 0.1% formic acid, and directly use it for LC-MS/MS analysis or peptide concentration measurement.
Product Notes
(1) The experimental operations should be conducted at low temperatures, such as at 4°C, to prevent modification loss.
(2) The lysis buffer used must be freshly prepared and contain appropriate inhibitors.
(3) After enrichment, samples can be buffer exchanged or concentrated using methods such as ultrafiltration or dialysis.
(4) Beads should be thoroughly mixed before use to avoid uneven sedimentation that may lead to fluctuating enrichment efficiency.
Features and Benefits
1. High Specificity and Efficiency
The lactate-lysine enrichment kit uses high-affinity lactylation binding antibodies or lactylation binding domains, which accurately recognize lactylated lysine residues. It effectively reduces non-specific binding and enhances the purity of target protein enrichment, providing clearer data for subsequent mass spectrometry analysis and immunological experiments.
2. High Recovery Rate
The optimized binding and elution systems ensure efficient capture of low-abundance lactylation-modified peptides, with excellent recovery capability. It is particularly suitable for deep exploration of lactylation modifications in complex samples and studies of low-expression sites.
3. Fast and Convenient
The process is streamlined and easy to follow, with the entire enrichment procedure completed within a day. No additional reagents or complex equipment are required, making it ideal for both conventional laboratory use and automated platforms.
4. Wide Sample Applicability
Lactate-lysine enrichment kit is suitable for a variety of sample sources, including mammalian cells and tissue homogenates. It supports common research models such as humans, mice, and rats, broadening experimental application scenarios.
Applications
1. Lactylation Proteomics Research
The lactate-lysine enrichment kit is designed for large-scale lactylation proteomics studies, helping researchers identify and quantify lactylated proteins, supporting the construction of lactylation modification maps.
2. Cell Metabolism and Energy Regulation Research
Lactylation is closely related to cellular metabolism. By enriching lactylated peptides, researchers can delve deeper into the role of lactylation in cellular energy metabolism, redox reactions, and other processes.
3. Low Abundance Lactylation Signal Mining
Lactylation modifications are often present at low abundance in cells or tissues. After enrichment using lactate-lysine enrichment kit, targeted mass spectrometry verification can significantly improve detection sensitivity, making it suitable for early diagnostic biomarker development and other research areas.
4. Immune Regulation and Inflammatory Response
Lactylation plays a crucial role in regulating immune cell activity. The enrichment of lactylated peptides can help to study the specific roles of lactylation in immune responses and inflammation.
5. Lactylation Modification Screening in Tumor Metabolism Research
In tumor cells, glycolytic activity is significantly enhanced, leading to a large accumulation of lactate that induces lactylation of various metabolic proteins. Using lactate-lysine enrichment kit to enrich its lactylated proteins can explore metabolic reprogramming mechanisms in the tumor microenvironment, uncover potential lactylation targets, and develop anti-tumor strategies.
FAQs
Q1: What Types of Samples Does the Kit Support?
A1: The lactate-lysine enrichment kit supports various sample types, including cells, tissues, and others, making it suitable for studies across different experimental backgrounds.
Q2: Is the Kit Compatible with Samples from Different Species?
A2: Yes, the kit has good cross-species compatibility and is suitable for samples from humans, mice, rats, and other species.
Q3: How Can the Purity of the Sample Be Ensured after Enrichment?
A3: Through optimized washing steps, non-specifically bound peptides are removed, ensuring that the enriched lactylated peptides are of high purity, making them suitable for downstream mass spectrometry analysis.
Q4: Is a De-lactylase Inhibitor Necessary to Use with the Lactate-Lysine Enrichment Kit?
A4: Yes, de-lactylase inhibitors are critical to maintaining the lactylation modification. To prevent lactylation proteins from being removed during the lysis process, it is recommended to add de-lactylase inhibitors to the lysis buffer to ensure the stability of the lactylation modification.
Q5: Does the Washing Buffer Provided in the Kit Need to Be Adjusted According to Experimental Requirements?
A5: Generally, the washing buffer provided is optimized and suitable for most sample types. However, under specific experimental conditions, such as for membrane proteins, it may be necessary to adjust the composition or pH of the washing buffer according to the characteristics of the target protein. It is recommended to perform a pre-experiment validation before conducting special experiments.
