background:

Trypsin belongs to the serine protease family and can specifically cleave the carboxyl groups of lysine and arginine residues in polypeptide chains. Trypsinogen is secreted by the pancreas and is decomposed into activated trypsin by the restriction of enterokinase or trypsin, which is an endopeptidase. It not only acts as a digestive enzyme, but also limits the decomposition of precursors of other enzymes such as chymotrypsinogen, carboxypeptidase, and phospholipase, and plays an activating role. It is the most specific protease.

Overview:

Rhinogen ^® Quick™ Trypsin (Sequencing Grade) is a recombinant trypsin obtained by recombinant expression in a recombinant E. coli system and purification by multi-step chromatography. The enzyme is methylated and mutated to ensure that it does not self-cleave, thereby removing the pseudo-chymotrypsin activity caused by self-cleavage. The purity of the enzyme is ≥95%, and there is no contamination by chymotrypsin and other proteases, and TPCK is not required to inhibit the activity of other enzymes. The amino acid sequence is homologous to porcine trypsin, with a molecular weight of approximately 25kD, and can specifically cleave the carboxyl groups of lysine and arginine residues in polypeptide chains.

Product Packaging:

Rhinogen ^® Quick™ Trypsin (Sequencing Grade) packaging specifications are as follows:

Supporting reagents:

Quick™ Trypsin Enzyme and Quick™ Reaction Enhancer are provided as lyophilized powders.

Note: Quick™ Reaction Enhancer for Rhinogen ^® Quick™ Trypsin (Sequencing Grade) can be purchased separately, Cat. No. EB17.

Product Source:

Rhinogen ^® Quick™ Trypsin (Sequencing Grade) is a recombinant trypsin produced by recombinant E. coli system and purified by multi-step chromatography. Its amino acid sequence is homologous to porcine trypsin sequence and its molecular weight is about 25kDa.

Product quality:

SDS-PAGE analysis showed purity ≥95%; no contaminating protease activity was detected.

Product Features:

Optimal reaction pH: 7.0~8.0.

Storage conditions:

Transported in ice packs. Immediately after receiving the product, place it at -20℃ and seal it to prevent moisture. After the enzyme is dissolved, store it at -70℃; if it is stored at 2-8℃ after dissolution, it must be used within 24 hours; after the Quick™ reaction enhancer lyophilized powder is dissolved, it can be stored at -70℃ for one month and at 2-8℃ for one week. After the enzyme solution and Quick™ reaction enhancer are dissolved, avoid leaving them at room temperature for a long time. They can be divided and packaged to avoid repeated freezing and thawing.

application:

1. Complete rapid digestion of antibodies or proteins within minutes;

2. Peptide spectrum , peptide fingerprint or protein sequence analysis;

3. Proteomics research;

4. Increase protein sequence coverage;

5. Improve the in-solution enzymatic hydrolysis scheme.

characteristic:

Rhinogen ^® Quick™ Trypsin (Sequencing Grade) is a highly purified and very stable recombinant protease with high stability and high specific activity. It is suitable for peptide mapping , peptide fingerprinting or protein sequence analysis in proteomics research.

1. High purity : SDS-PAGE analysis, purity ≥95%;

2. High consistency : Each batch of products undergoes strict quality control to achieve batch-to-batch stability ;

3. No animal origin : Recombinant production, no exogenous virus contamination, and no animal origin raw materials are used in the production process;

4. Ultra-high efficiency : It can significantly shorten the protein enzymatic hydrolysis time, complete all enzymatic hydrolysis work within the same day, and is compatible with liquid chromatography (LC) and MS analysis.

Instructions:

1. The usage of trypsin is trypsin: target protein = 1:20~1:100 (w/w), and the optimal pH is 7.0~8.0;

2. The reaction volume can be scaled up or down;

3. Adding 1mM CaCl _{2 to the enzyme digestion reaction system} can increase the activity of trypsin;

4. After the reaction is completed, take a small portion of the reaction system for SDS-PAGE or RP-HPLC detection to confirm the enzyme digestion effect. The remaining reaction system is placed in an ice bath or frozen . If the enzyme digestion is incomplete, take out the reaction system and continue the reaction at 37°C;

5. For different protein samples, it is necessary to experiment to find the most suitable enzyme concentration and reaction time. The enzyme solution can be diluted with 50mM NH ₄ HCO ₃ or pH 7.0-8.0 buffer solution ;

6. This product is for research use only and is not intended for diagnosis or treatment of humans or animals.

Frequently asked questions

Q1: Is it possible to adjust the sample pretreatment temperature?

A1: Add Quick ^TM High temperature incubation after the working reagent is conducive to the stretching of the substrate, thereby exposing more cleavage sites. For hydrophobic proteins, you can increase the incubation temperature to 90°C or close to 100°C, but pay attention to whether high temperature incubation will produce precipitation. When precipitation occurs during the incubation of your substrate, you can choose to appropriately lower the incubation temperature. Of course, increasing the concentration of the reaction enhancer can also effectively alleviate the protein precipitation problem.

Q2: What should I do if complex proteins cannot be digested effectively and quickly according to the instructions?

The concentration of the rapid cutting reagent recommended in our instructions is suitable for rapid digestion of most proteins. For complex proteins (such as hydrophobic proteins such as membrane proteins) that cannot be effectively and completely cut according to the above operation, you can try to increase the concentration of the rapid cutting reagent according to the table below .

At the same time, you can also try to increase the pretreatment temperature, reduce the substrate concentration, increase the enzyme dosage and extend the reaction time.

Q3: Hydrolyzed Quick ^TM What is the situation when the peptide coverage detected after the reaction enhancer is low?

A3: When using trypsin to digest hydrophobic proteins such as membrane proteins, highly hydrophobic peptides or proteins may co-precipitate with the degradation products of Quick ^TM Reaction Enhancer, resulting in a lower coverage of peptides in the aqueous layer. If you think this is a problem, reduce the amount of Quick ^{TM Use a} lower ^{concentration} of the reaction enhancer (e.g. 0.5× or 0.25×) and avoid acidification. The reaction enhancer will not interfere with LC/MS and MALDI-MS analysis. In the meantime, you can try using aqueous solution containing isopropanol to dissolve the precipitate, extract the hydrophobic peptides and perform LC/MS analysis.