In-Gel Tryptic Digestion Kit

Product Details	Size	Storage Conditions
Coomassie Destaining Solution	50 mL	4℃
Silver Stain Destaining Solution A	20 mL
Silver Stain Destaining Solution B	20 mL
50 mmol/L NH4HCO3	30 mL
DTT（1M）	0.5 mL	-20℃
IAM（1M）	1 mL
Trypsin	25 μg/vial; 3 vials
Trypsin Dilution Buffer	5 mL	4℃
Peptide Extraction Buffer	10 mL

 

Protocol

The in-gel tryptic digestion kit features an intuitive and standardized workflow, allowing researchers to quickly master and execute the protocol. The main steps include:

1. Gel Excision and Destaining

(1) Place the SDS-PAGE gel in a clean glass dish. Excise the target protein bands and cut them into ~1 mm³ gel pieces. Transfer the gel pieces into an EP tube.

(2) Destaining Coomassie-stained samples:

Add 5-10 volumes of destaining solution relative to the gel volume (e.g., add 500-1000 µL of destaining solution to 100 µL gel pieces). Shake at 800 rpm at room temperature for 5 min. Discard the supernatant and repeat the destaining step 2-3 times until the blue color disappears. Alternatively, incubate statically at 4°C overnight.

(3) Destaining silver-stained samples:

a. Mix silver stain destaining solution A and B at a 1:1 volume ratio (prepare freshly before use). Add an equal volume of the mixed solution to the gel pieces (e.g., add 100 µL of destaining solution to 100 µL gel pieces). Pipette up and down repeatedly until the brown color disappears.

b. Immediately add 2 volumes of pure water (e.g., add 200 µL pure water to 100 µL gel pieces) to terminate the reaction. Let stand for 10 min, then remove the supernatant.

c. Add 0.5 volumes of 50 mmol/L NH₄HCO₃ to the gel pieces. Let stand for 20 min, then remove the supernatant.

  

2. Dehydration

Add 5-10 volumes of acetonitrile to the gel pieces. Let stand at room temperature until the gel pieces turn white and shrink into pellets. Discard the acetonitrile and leave the tube at room temperature to allow residual acetonitrile to evaporate completely.

Note: If the gel volume is large, repeat the acetonitrile wash as needed to ensure thorough dehydration.

  

3. Reduction and Alkylation

(1) Add 10 mmol/L DTT solution to the gel pieces. Add just enough volume to fully swell the gel pieces. Incubate in a 56°C water bath for 1 hour. Briefly centrifuge and discard the excess solution.

(2) Add an equal volume of 20 mmol/L IAM solution to the gel pieces. React at room temperature in the dark for 1 hour. Briefly centrifuge and discard the excess solution.

(3) Add 5-10 volumes of coomassie destaining solution to the gel pieces. Vortex briefly, centrifuge, and discard the supernatant.

(4) Add 5-10 volumes of acetonitrile. Let stand at room temperature until the gel pieces turn white and shrink into pellets. Discard the acetonitrile and allow the remaining solvent to evaporate completely at room temperature.

 

4. Tryptic Digestion

Add trypsin (concentration: 0.025 μg/μL) to the dried gel pieces. One microgram of trypsin can digest approximately 50 μg of protein. Incubate at 37°C in a constant-temperature incubator for about 16 hours.

Note: If the gel volume is large, determine the required amount of trypsin (in μg), then dilute the 0.025 μg/μL trypsin with 50 mmol/L NH₄HCO₃ to a volume sufficient to fully swell the gel pieces before adding it to the dried gel.

 

5. Peptide Extraction

(1) Add 2-3 volumes of peptide extraction solution to the gel pieces and incubate at 37°C for 1 hour.

(2) Sonicate for 5 minutes, then briefly centrifuge. Transfer the supernatant to a new EP tube. Repeat the extraction once.

(3) Combine the extraction solutions and vacuum-dry at 57°C.

 

Features and Benefits

1. Ready-to-Use

All buffers are pre-formulated, eliminating complex preparation steps and improving workflow efficiency.

  

2. Comprehensive

Contains all reagents needed for gel destaining, reduction, alkylation, and tryptic digestion of proteins.

 

3. Robust

Optimized protocols and reagents ensure reliable digestion and consistent data across varied conditions.

 

4. Broad Compatibility

Compatible with a wide range of mass spectrometry platforms, meeting diverse analytical requirements.

 

5. High Specificity

Includes highly purified, MS-grade modified trypsin with no chymotrypsin activity and minimal autolysis.

 

Applications

The in-gel tryptic digestion kit is suitable for a wide range of protein research projects, especially in the following contexts:

1. Protein Identification

Enables in-gel digestion of isolated target proteins for sequence information acquisition via mass spectrometry.

 

2. Post-Translational Modification (PTM) Analysis

Provides reliable and accurate peptide digestion results for identifying modification sites such as phosphorylation and acetylation.

 

3. Protein Complex Analysis

Facilitates subunit-level digestion of separated protein complexes to investigate their composition and interactions.

 

4. Comparative Proteomics

Supports analysis of differentially expressed protein bands, aiding in disease mechanism research and drug target discovery.