Ubiquitination is an important form of post-translational modification that is widely present in eukaryotic cells. It plays a critical role in regulating protein degradation, signal transduction, cell cycle, DNA repair, immune response, and various other biological processes. With the advancement of proteomics technologies, studying protein ubiquitination has become a key approach to uncovering cellular physiological functions and disease mechanisms. However, due to the low abundance, transient nature, and structural diversity of ubiquitination modifications, direct detection in complex samples remains highly challenging. Efficient enrichment of ubiquitinated proteins or peptides from samples is a prerequisite for achieving high-sensitivity mass spectrometry analysis or specific antibody detection.
Traditional enrichment methods often suffer from high background, poor selectivity, and low reproducibility, making them insufficient for the demands of high-throughput and high-sensitivity modification studies. The ubiquitin enrichment kit employs anti-K-ε-GG antibody materials to achieve precise capture of ubiquitinated proteins or peptides, with high specificity, a simplified workflow, and short operation times, making it widely applicable to mass spectrometry analysis, immunological research, and studies on ubiquitin-mediated regulatory mechanisms. It is an ideal pretreatment tool for conducting ubiquitination modification research.
Product Overview
The ubiquitin enrichment kit from MtoZ Biolabs is based on the capture principle of anti-K-ε-GG (Lys-Gly-Gly) specific antibodies. It is suitable for peptides derived from a variety of fresh or frozen tissues, adherent or suspension cells, and expression system products. The kit includes optimized wash, binding, and elution buffers to ensure a gentle and efficient enrichment process with high sample recovery.
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 ubiquitin enrichment kit adopts the capture principle of anti-K-ε-GG (Lys-Gly-Gly) specific antibodies, and the entire workflow is simple and suitable for different laboratory environments and experimental scales. The following are the recommended standardized steps.
Peptide-Level Ubiquitin Enrichment Workflow:
1. Sample Preparation
(1) Perform desalting of digested protein samples using a C18 column or an equivalent method, then lyophilize.
(2) It is recommended to start with >500 µg of digested protein, and reconstitute the peptides in Wash Buffer 2, ensuring complete dissolution.
2. Antibody Coupling to Beads
(1) Wash the beads with Wash Buffer 1, repeat twice.
(2) Resuspend the beads in 200 µL Wash Buffer 1, add an appropriate amount of antibody, and rotate at 4°C for 2 hours.
(3) Centrifuge to separate the beads and retain the supernatant.
Note: It is recommended to quantify the supernatant after incubation using a BCA assay to ensure complete antibody binding.
3. Peptide Incubation
(1) Add the reconstituted peptides to the bead pellet and rotate at 4°C overnight.
4. Washing to Remove Impurities
(1) Centrifuge to separate the beads.
Note: Users can decide whether to retain the supernatant containing non-ubiquitinated peptides based on their experimental needs.
(2) Wash the beads twice with Wash Buffer 2, retaining the beads each time.
(3) Wash the beads twice more with Wash Buffer 3, retaining the beads.
5. Elution of Ubiquitinated Peptides
(1) Transfer the beads to a new collection tube, add 100 µL Elution Buffer, incubate at room temperature for 10 minutes, centrifuge to collect the supernatant; then add another 100 µL Elution Buffer, repeat the process, and collect a total of 200 µL.
(2) Desalt the enriched ubiquitinated peptide samples using a C18 column or an equivalent method, then lyophilize.
6. Downstream Analysis
Reconstitute the dried peptides with 20 µL 0.1% formic acid and proceed directly to LC-MS/MS analysis or peptide concentration determination.
Notes
(1) Perform all procedures at low temperature (e.g., on ice) to maintain the stability of ubiquitination modifications.
(2) Pre-cool all buffers before use.
Features and Benefits
1. High Selectivity and Specificity
Utilizing high-affinity antibodies, the ubiquitin enrichment kit enables highly selective recognition of ubiquitination modification sites, achieving precise enrichment of ubiquitinated proteins or peptides. This effectively removes non-specific background signals, ensuring accuracy and reproducibility of subsequent analyses.
2. Multiple Enrichment Strategies Available
Offers peptide-level enrichment options to accommodate different research purposes, such as studying ubiquitination regulatory mechanisms and site-specific analysis. Flexible adaptation to various experimental needs and research depths is supported.
3. Easy Operation
Provides complete sets of key reagents and instructions for sample preparation, binding, washing, and elution. The entire enrichment workflow can be completed within two days without the need for additional reagents or complex equipment, making it suitable for routine laboratories and automated platforms.
4. Broad Sample Compatibility
Compatible with a wide range of sample types, including adherent/suspension cells, tissues, recombinant expression products, and biological fluids. Applicable across multiple species such as human, mouse, and rat backgrounds.
5. Suitable for Various Downstream Analyses
The enriched products are compatible with mass spectrometry, Western blotting, ELISA, immunoprecipitation, and other downstream applications, offering strong versatility and potential for experimental expansion.
Applications
1. Ubiquitination Site Analysis
Through peptide-level enrichment strategies combined with LC-MS/MS platforms, high-throughput identification and quantification of K-ε-GG-modified peptides can be achieved. This enables detailed mapping of ubiquitination site distribution, modification patterns, and dynamic changes, supporting the construction of comprehensive ubiquitin modification atlases.
2. Signaling Pathway and Degradation Mechanism Studies
By enriching ubiquitinated signaling molecules, researchers can investigate the functional roles of ubiquitination regulation within classical signaling pathways.
3. Drug Screening and Functional Validation
In combination with compound intervention experiments, changes in protein ubiquitination levels can be monitored to evaluate the effects of drugs on protein degradation or ubiquitination regulation.
4. Disease Mechanism Research
FAQs
Q1: What Types of Ubiquitination Can Be Detected Using the Kit?
A1: The ubiquitin enrichment kit can detect various forms of ubiquitination, including mono-ubiquitination and polyubiquitin chains (K48/K63). Some products are specifically designed for site-level enrichment of K-ε-GG peptides.
Q2: Should Deubiquitinase Inhibitors Be Added to the Sample?
A2: It is strongly recommended to add deubiquitinase inhibitors during sample preparation to prevent the removal of ubiquitin modifications and ensure the authenticity of the enrichment results.
Q3: Can the Enriched Samples Be Used for Mass Spectrometry Analysis?
A3: Absolutely. The enriched products are fully compatible with LC-MS/MS platforms and are suitable for high-throughput identification and quantification of ubiquitination sites.
Q4: Is the Kit Applicable to All Species?
A4: The kit is applicable to most mammalian samples, including human, mouse, and rat. For other species, small-scale pilot testing is recommended before large-scale application.
