Trypsin

Manufactured by Merck Group

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Trypsin is a serine protease enzyme that is commonly used in cell biology and biochemistry laboratories. Its primary function is to facilitate the dissociation and disaggregation of adherent cells, allowing for the passive release of cells from a surface or substrate. Trypsin is widely utilized in various cell culture applications, such as subculturing and passaging of adherent cell lines.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (406 mentions) Fetal bovine serum (fbs), by Merck Group (201 mentions) Ethylene diamine tetraacetic acid (edta), by Merck Group (196 mentions) Pepsin, by Merck Group (181 mentions) Penicillin, by Merck Group (166 mentions) Streptomycin, by Merck Group (155 mentions) Penicillin streptomycin, by Merck Group (144 mentions) Dimethyl sulfoxide (dmso), by Merck Group (144 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (143 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (133 mentions) Streptomycin, by Thermo Fisher Scientific (123 mentions) Penicillin, by Thermo Fisher Scientific (116 mentions) Collagenase, by Merck Group (113 mentions) L glutamine, by Merck Group (106 mentions) Phosphate buffered saline (pbs), by Merck Group (105 mentions) Chymotrypsin, by Merck Group (102 mentions) Bovine serum albumin (bsa), by Merck Group (97 mentions) Dithiothreitol (dtt), by Merck Group (84 mentions) Dulbecco modified eagle medium (dmem), by Merck Group (79 mentions) Dnase, by Merck Group (77 mentions)

Spelling variants of this product

SOLu-Trypsin (8 citations) Trypsin Singles (5 citations) Trypsin 1X (4 citations) L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (3 citations) Trypsin/PBS (3 citations) Trypsin Single Proteomics Grade Kit (2 citations) Mass spectrometry-grade trypsin (2 citations) Trypsin type XIII (2 citations) Trypsin and chymotrypsin (2 citations) TPCL-trypsin (2 citations)

3 740 protocols using trypsin

Trypsin Sensitivity of Synaptic Vesicle Proteins

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AGO2/synaptophysin Westerns: SVs were isolated and concentrated as described above. One preparation of vesicles was split into 10 equal samples. Each sample consisted of 5 μl of SVs (6 mg/ml) in PBS, pH 7.4. Paired samples were treating in the following manner: no Trypsin treatment, 2 μl Trypsin (T1426, 10 mg/ml; Sigma-Aldrich) treated for 5 mins, 10 mins, 20 mins or 30 mins. Westerns were conducted using antibodies against synaptophysin or AGO2.
SV RNA sensitivity to Trypsin: SVs were isolated and concentrated as described above. For each assay, 1 preparation of vesicles was utilized, and split into 4 equal samples of 50 μl. Each sample consisted of SVs (6 mg/ml) in PBS (80 mM Na₂HPO₄ and 25 mM NaH₂PO₄, 100 mM NaCl), pH 7.4. The 4 parallel treatments conducted on the SV preparations were: 1) addition of 50 μl PBS, pH 7.4, 2) addition of 50 μl PBS pH 7.4 and 0.5 μl Trypsin (T1426,10 mg/ml; Sigma-Aldrich) for 20 minutes, 3) addition of 50 μl PBS pH 7.4 and 0.5 μl Trypsin for 20 min, followed by 5 μl Trypsin Inhibitor (T9128, 6.5 mg/10 ml, Sigma-Aldrich) and 1 μl RNAse for 30 min., 4) addition of 50 μl combined Trypsin, 200 mM DM for 20 min, followed by 5 μl Trypsin Inhibitor and RNAse treatment for 30 minutes. RNA was extracted after the treatments and quantified as described above.

Li H., Wu C., Aramayo R., Sachs M.S, & Harlow M.L. (2015). Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA). Scientific Reports, 5, 14918.

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LDL Modification and Oxidation Protocols

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Human plasma LDL (Lee Biosciences) was modified using a previously established method.³⁸ (link) The LDL protein concentration was measured using the Lowry Protein Assay (Thermo Fisher Scientific). For eLDL modification, based on the protein concentration, the LDL solution was incubated at 37 °C with different amounts of 0.05% trypsin (Sigma-Aldrich) and cholesterol esterase [Sigma-Aldrich, reconstituted to 1 mg/ml using 0.4 M potassium phosphate monobasic (Sigma-Aldrich)]. Briefly, LDL was first incubated with 7 μg trypsin/mg LDL protein for 6 h, then 12 μg cholesterol esterase/mg LDL protein for 10 h, then trypsin again at 24 μg/mg LDL protein for 6 h, and finally 29 μg cholesterol esterase/mg LDL protein for 48 h. Following the incubations, the LDL solution was dialyzed against PBS for 20–24 h.
For oxLDL modification, 5 mg of LDL was incubated in a 1 ml 10 μM CuSO₄ solution at 37 °C for 48 h. Following incubation, the modified LDL solution was dialyzed against PBS for 20–24 h.
TNF-α (Sigma-Aldrich or PeproTech) was reconstituted at 10 μg/ml in sterile PBS, aliquoted, and frozen at −20 °C. Aliquots were used within 6 months of reconstitution.

Lee J.H., Shores K.L., Breithaupt J.J., Lee C.S., Fodera D.M., Kwon J.B., Ettyreddy A.R., Myers K.M., Evison B.J., Suchowerska A.K., Gersbach C.A., Leong K.W, & Truskey G.A. (2023). PCSK9 activation promotes early atherosclerosis in a vascular microphysiological system. APL Bioengineering, 7(4), 046103.

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Enzymatic Degradation of Cartilage Specimens

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Enzymatic degradation of specimens (n = 36) was conducted using either collagenase or trypsin. Collagenase D (0.1 mg/ml, Sigma-Aldrich Inc., St. Louis, MO, USA) was utilized for digestion of the collagen network, whereas trypsin (0.5 mg/ml, T4299, Sigma-Aldrich Inc., St. Louis, MO, USA) was used to deplete the proteoglycans from the extracellular matrix. 27, 28 Collagenases D explicitly targets and cleaves the collagen molecules in the triple helix region, whereas trypsin cuts the peptide bond on the C-terminal side of lysine and arginine amino acids (Sigma-Aldrich Inc).
Prior to enzymatic degradation, the specimens were subdivided into three groups: collagenase 24 h (C24, n = 12), collagenase 90 min (C90, n = 12), and trypsin 30 min (T30, n = 12). Specimens were incubated at 37 C and 5% CO 2 in PBS solution containing the respective enzymes with supplementary antibiotics (penicillin-streptomycin-amphotericin B, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B, Sigma-Aldrich Inc., St. Louis, MO, USA). 29 For mild cartilage degradation, we applied a shorter incubation time of 30 min for trypsin (T30) and 90 min for collagenase (C90). A long incubation time of 24 h was also applied with collagenase (C24) to induce severe damage. After enzymatic treatment, specimens were rinsed in PBS before the Raman spectroscopic measurements.

Shaikh R., Nippolainen E., Virtanen V., Torniainen J., Rieppo L., Saarakkala S., Afara I.O., & Töyräs J. (2021). Raman spectroscopy is sensitive to biochemical changes related to various cartilage injuries. Journal of Raman Spectroscopy, 52(4), 796-804.

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Enzymatic Cartilage Degradation Protocol

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Two enzymes were used for degradation of the samples in group 2. Collagenase D (Sigma Aldrich) was used for the degradation of the collagen network,35 (link) while trypsin (T4299, Sigma Aldrich) was used for proteoglycan digestion (with minor collateral effect on the collagen network).17 (link) The samples in group 2 were further divided into three subgroups according to the enzyme and duration of treatment as follows: collagenase 24 h (E1, n = 12), collagenase 90 min (E2, n = 12) and trypsin 30 min (E3, n = 12). The prewarmed samples were incubated at 37 °C and 5% CO₂ in PBS solution containing the respective enzymes (0.1 mg/mL for Collagenase D, and 0.5 mg/mL for trypsin) and supplemented with antibiotics, including Penicillin–Streptomycin–Amphotericin B (100 U/mL Penicillin, 100 µg/mL Streptomycin and 0.25 µg/mL Amphotericin B, stabilized, Sigma-Aldrich) for the different times specified previously. The incubation times of 30 min for trypsin and 90 min for collagenase were applied to induce mild cartilage degradation that mimic early OA. In order to induce severe damage, long incubation time of 24 h in collagenase was applied.

Nippolainen E., Shaikh R., Virtanen V., Rieppo L., Saarakkala S., Töyräs J, & Afara I.O. (2020). Near Infrared Spectroscopy Enables Differentiation of Mechanically and Enzymatically Induced Cartilage Injuries. Annals of Biomedical Engineering, 48(9), 2343-2353.

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Tryptic Digestion of Membrane Protein

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Trypsin (Sigma-Aldrich) was added at a Trypsin:MtrA ratio of 1:2,000 (w/w) to a solution of refolded cobalamin-bound soluble domain of membrane-associated MtrA from M. jannaschii in the presence of 10 mM CaCl₂ (final concentration) and incubated for 6 min at 30 °C. Proteolysis was stopped by adding the irreversible Trypsin inhibitor, N_α-tosyl-l-lysine chloromethylketone (0.2 mM; Sigma-Aldrich). The cleaved protein was injected onto a HiPrep Sephacryl S100 16/60 column equilibrated with GF buffer. The eluted protein was analysed by SDS-PAGE.

Wagner T., Ermler U, & Shima S. (2016). MtrA of the sodium ion pumping methyltransferase binds cobalamin in a unique mode. Scientific Reports, 6, 28226.

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LDL Modification and Oxidation Protocols

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Protein Digestion and Labeling for Quantitative Proteomics

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Proteins (100 μg) were digested with trypsin (protein:trypsin = 20:1; Sigma) and incubated at 37 °C for 4 h. More trypsin was then added at the above proportion and incubation continued at 37 °C for 8 h. The peptides were then dried with a vacuum centrifugal pump and 0.5 M TEAB was used to resolubilize the peptides. Peptides were then labeled with iTRAQ with incubation at room temperature for 2 h according to the manufacturer’s instructions. The labeled peptides were mixed and separated on an SCX column through the LC-20AB liquid phase system (Shimadzu, Kyoto, JPN).

Wang H., Zhu X., Shen J., Zhao E.F., He D., Shen H., Liu H, & Zhou Y. (2019). Quantitative iTRAQ-based proteomic analysis of differentially expressed proteins in aging in human and monkey. BMC Genomics, 20, 725.

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Evaluating Probiotic Tolerance to Digestive Conditions

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LAB isolates were cultured in MRS broth at 37°C for 24 h and subcultured into fresh MRS broth and incubated for another 24 h. MRS media with trypsin (Sigma, Shanghai, China) at concentrations of 8, 10, 12, 14, and 16 g/L were prepared. Samples of the 3rd subcultures of LABs (2 μL each, corresponding to 6 x 10 8 CFU/mL) were spotted onto MRS-trypsin medium, incubated for 24 h at 37°C, and detected at OD 600 .
Tolerance to pepsin (Sigma, Shanghai, China) was determined as per the procedure used for trypsin tolerance. MRS medium containing 37% hydrochloric acid (wt/v) (Guoyao, Shanghai, China), pH 3.0, was prepared and 5 mg/mL pepsin (NF 3000 U/mg) was added to simulate gastric juice. MRS medium without pepsin, pH 6.25, was used as control. Samples of the 3rd subculture of LABs (2 μL each, corresponding to 6 x 10 8 CFU/mL) were spotted onto MRS-pepsin medium and incubated for 24 h at 37°C and detected at OD 600 .

Xie Z.L., Bai D.P., Xie L.N., Zhang W.N., Huang X.H, & Huang Y.F. (2015). Intestinal lactic acid bacteria from Muscovy duck as potential probiotics that alter adhesion factor gene expression. Genetics and molecular research : GMR, 14(4).

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Chloroplast Protein Import Assay

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Protein import assay was done using intact chloroplasts isolated from 10- to 13-day-old pea seedlings and [³⁵S]-labeled proteins as previously described (Inoue and Potter, 2004 (link); Inoue et al., 2006 (link)). The radiolabeled proteins were synthesized using T_NT^® coupled reticulocyte lysate system (Promega) and T7 (for Toc75-IV and Tic22), T3 (for OEP80tr), SP6 (for Toc75, and OEP80) RNA polymerases with [³⁵S]Met (Perkin Elmer, Waltham, MA). Post-import fractionation of chloroplasts was done as described (Inoue et al., 2006 (link)). For post-import protease treatment, the chloroplasts containing the imported proteins were treated with thermolysin or trypsin (both are from Sigma-Aldrich Corp, St. Louis, MO) at a 1:1 mass ratio with the amount of chlorophylls incubated in the import reaction in import buffer with (for thermolysin) or without (for trypsin) 1 mM CaCl₂, respectively, for 30 min in the dark on ice (for thermolysin) or at room temperature (for trypsin). The protease reactions were quenched by adding EDTA to the final concentration of 5 mM (for thermolysin) or trypsin inhibitor at a 10:1 mass ratio of the inhibitor to the protease (for trypsin) in import buffer. For the energy-dependency assay, the reaction was done using translation products pre-treated with 50 U/mL apyrase (Sigma-Aldrich) at room temperature for 15 min.

Day P.M., Potter D, & Inoue K. (2014). Evolution and targeting of Omp85 homologs in the chloroplast outer envelope membrane. Frontiers in Plant Science, 5, 535.

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Protein Hydrolysis and Tryptic Digestion

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Protein samples were hydrolyzed using trypsin (Sigma-Aldrich, St. Louis, MO, USA, T8658), an enzyme present in the gastrointestinal tract of mammals, to digest the protein. Extracted protein samples were mixed with 20% TCA and allowed to precipitate for 2 h at 4 °C. The mixture was then centrifuged at 4500 rpm for 5 min, and the supernatant was discarded. The precipitate was washed 2 times with pre-chilled acetone. The pellets were then dried and triethylammonium bicarbonate (TEAB) (Sigma-Aldrich, 241059) was added to obtain a final concentration of 200 mM. trypsin was added at a ratio of 1:50 (protease:protein m/m) and allowed to hydrolyze overnight. After digestion with trypsin, dithiothreitol (DTT) (Sigma-Aldrich, 43815) was added to obtain a final concentration of 5 mM and then reduced for 30 min at 56 °C. Finally, iodoacetamide was added to obtain a final concentration of 11 mM and incubated for 15 min at room temperature after covering it with silver paper (to avoid light).

Ali M.A., Qin Z., Dou S., Huang A., Wang Y., Yuan X., Zhang Y., Ni Q., Azmat R, & Zeng C. (2023). Cryopreservation Induces Acetylation of Metabolism-Related Proteins in Boar Sperm. International Journal of Molecular Sciences, 24(13), 10983.

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