Proteomics research relies heavily on efficient sample preparation workflows, as the quality of sample preparation directly impacts the accuracy of downstream analyses. This is especially critical in experiments involving protein-protein interaction studies, post-translational modification (PTM) analysis, and the identification of low-abundance proteins, where digestion efficiency and recovery rate are key factors. Traditional protein digestion methods primarily include in-solution digestion and in-gel digestion. In-solution digestion is suitable for relatively purified protein solutions; however, in complex samples such as cell lysates or immunoprecipitation-enriched proteins, the presence of high concentrations of detergents and salts may interfere with enzyme activity, leading to incomplete digestion or reduced efficiency. In contrast, in-gel digestion is commonly used for protein identification following SDS-PAGE separation. While this method can reduce interference, it typically results in lower protein recovery, particularly for low-abundance proteins, where significant sample loss can occur.
To improve protein digestion efficiency, reduce nonspecific interference, and enhance sample recovery, magnetic bead-based on-beads digestion technology has gained increasing attention in recent years. This method is primarily applied in protein-protein interaction studies (such as co-immunoprecipitation, or Co-IP, and pull-down assays), tag-based protein enrichment (e.g., His-tag/Ni-NTA purification), and enrichment of low-abundance proteins. Compared with traditional digestion methods, on-beads digestion enables proteolysis to occur directly on the surface of magnetic beads, thereby minimizing sample loss during transfer and improving the sensitivity of low-abundance protein detection. In addition, this approach effectively reduces background contamination, such as unbound high-abundance proteins or buffer components, resulting in more accurate and reliable downstream mass spectrometry analysis.
Product Overview
Based on the advantages of magnetic bead-based protein digestion technology, MtoZ Biolabs has developed the on beads protein digestion kit which designed to provide researchers with an efficient, stable, and user-friendly protein digestion solution. The kit is specifically optimized for protein samples bound to magnetic beads and enables direct proteolysis on the bead surface without the need for additional elution steps, thereby minimizing sample loss and improving the detection sensitivity of low-abundance proteins. By using an optimized buffer system that mitigates the negative effects of residual high salt and detergents on enzymatic activity, enhancing digestion efficiency and ensuring high reproducibility and consistency across experiments. It is particularly well-suited for applications such as co-immunoprecipitation (Co-IP), tagged protein enrichment (His-tag/Ni-NTA), and protein complex characterization. The resulting digested samples are fully compatible with high-sensitivity downstream analyses, including liquid chromatography-mass spectrometry (LC-MS/MS), making the on beads protein digestion kit an ideal tool for accurate and comprehensive protein identification.
Product Details
|
Product Details |
Concentration / Quantity |
Size |
Storage Conditions
|
|
Beads A |
50 μg/μL |
5 mL |
4℃ |
|
Beads B |
50 μg/μL |
5 mL |
|
|
Bead Incubation Buffer |
1× |
30 mL |
|
|
Bead Washing Buffer |
1× |
500 mL |
Protocol
The on beads protein digestion kit employs an optimized magnetic bead–based digestion workflow, streamlining experimental procedures and minimizing protein loss. The operation steps are as follows:
1. Sample Requirements After Pretreatment
Total protein amount ≥ 50 μg, protein concentration ≥ 1 μg/μL, proteins must be pre-reduced and alkylated.
2. Beads Washing
a. Mix Beads A and Beads B at a 1:1 ratio. Gently vortex for 30 seconds to mix thoroughly. Place the tube on a magnetic rack and allow it to stand for 2 minutes. Discard the supernatant.
b. Add 1 mL ultrapure water, gently vortex for 30 seconds, place on the magnetic rack, let stand for 2 minutes, and discard the supernatant. Repeat this washing step three times.
c. Finally, resuspend the beads in ultrapure water to a concentration of 100 μg/μL for storage at 4°C.
3. SP3 Enrichment
Taking 100 μg protein as an example, add Beads and magnetic bead incubation solution according to the table below. The actual experiment can increase or decrease in proportion. After addition, bonding was 900 rpm at room temperature (24°C) for 15 minutes.
|
Sample Reaction Volume |
Beads Volume |
Bead Incubation Buffer |
|
100 μL |
10 μL |
110 μL |
4. SP3 Washing
a. Briefly centrifuge the sample and place it on a magnetic rack. Let it stand for 2 minutes, then transfer the supernatant to a new tube using a pipette. The supernatant can be stored at -80°C.
b. Add 500 μL SP3 Wash Buffer to the beads, pipette to mix thoroughly, then place the sample on the magnetic rack for 2 minutes and discard the supernatant. Repeat the washing step a total of 3 times. After the final wash, carefully remove all wash buffer and air dry the beads at room temperature for 20-30 minutes.
Note: Dry only until no shiny liquid remains on the bead surface. Do not over-dry.
5. SP3 Digestion
a. Add 300 μL of digestion buffer (choose buffer type according to the protease used), vortex at 1,000 rpm for 5 minutes at room temperature to fully resuspend the beads.
b. Add the appropriate amount of protease based on the desired enzyme-to-substrate ratio. Digest overnight at 1,000 rpm for 14-18 hours.
6. Collection of Digested Peptides
Briefly centrifuge the sample and place it on a magnetic rack. Let it stand for 2 minutes, then transfer the digestion supernatant to a new tube using a pipette. The collected digestion solution can be subjected to optional purification, such as C18 column enrichment or filtration, before downstream analysis.
Applications
1. Immunoprecipitation (Co-IP) Protein Identification
Suitable for on-bead digestion in Co-IP experiments, enhancing the identification efficiency of interacting proteins.
2. Affinity-Purified Protein Analysis
Applicable to protein samples purified by His-tag, GST-tag, or Ni-NTA affinity methods, minimizing protein loss caused by additional elution steps.
3. Post-Translational Modification (PTM) Protein Detection
Can be used for samples enriched for phosphorylation, acetylation, ubiquitination, and other PTMs to ensure efficient digestion and improve the accuracy of modification site identification.
FAQs
Q1: What Are the Advantages of the On Beads Protein Digestion Kit Compared to Traditional in-Solution Digestion?
A1: Traditional in-solution digestion can lead to the loss of low-abundance proteins and may introduce additional background interference during elution. The on beads protein digestion kit from MtoZ Biolabs enables proteins to be digested directly on the surface of magnetic beads without additional elution steps, significantly reducing sample loss and ensuring efficient, precise digestion. It is especially suitable for protein–protein interaction studies, low-abundance protein detection, and post-translational modification (PTM) analysis.
Q2: What Types of Bead-Bound Protein Samples Are Compatible with the Kit?
A2: The on beads protein digestion kit is compatible with a wide range of magnetic bead-based workflows, including co-immunoprecipitation (Co-IP), affinity purification (His-tag/Ni-NTA), GST-tag, streptavidin-biotin systems, and other tagged protein enrichment methods. Whether for studying protein interactions or analyzing protein complexes, the kit ensures highly efficient digestion and excellent compatibility with mass spectrometry.
Q3: Is the Kit Compatible with LC-MS/MS Analysis?
A3: Yes, The on beads protein digestion kit is specifically designed for high-sensitivity proteomics applications. It utilizes optimized digestion buffers and high-purity proteases to ensure the digested peptides are directly compatible with LC-MS/MS. Extensive validation shows that the method significantly enhances protein identification coverage and data consistency, making it an ideal solution for high-throughput proteomic research.
