Trypsin solution preparation

Prepare a solution of TCPK-trypsin in 50 mM ammonium bicarbonate, pH 8.0, at a concentration of 100 ng/pl. Add 10 pi of the trypsin solution to every 10 pg of combined, labeled protein solution from Step 6. Incubate at 37°C for 12-16 hours or overnight with mixing. 

This may be achieved by complete immersion of full-thickness skin in trypsin solution or by placing the heat-separated epidermis for 24 h at 37°C on a filter paper soaked with the enzyme preparation [64, 83]. Other techniques, such as vacuum or chemically induced blistering, stretching, application of staphylococcal exfoliatin, or proteolytic digestion of viable cells, are seldom reported [74, 87-89],... 

Tissue Preparation 1. P2 Sprague-Dawley Rat pups. 2. Dulbecco Modified Eagle s Medium (DMEM), high glucose, no glutamine, no pyruvate (Cambrex). 3. Ca +-Mg free Hanks Balanced Salt Solution (Cambrex). 4. 2.5% trypsin solution (Cambrex) diluted 1 10 in HBSS for 0.25% final, aliquot and store at -20°C, stable for 1 year. 5. DNAse 1 (Boehringer Mannheim) prepare 1 mg/mL stock in PBS aliquot store at -20°C stable for 1 year. 6. Heat-inactivated FBS (Cambrex) aliquot and store at -20°C. 

Trypsin solution 1 mg/mL mg trypsin dissolved (Promega, Madison, WI, USA) in 20 mM ammonium carbonate (freshly prepared). 

The stock of 5% by weight trypsin solution consisted of the enzyme dissolved in 0.04M Tris-HCl buffer (pH=8.1) containing O.OIM CaCl2 (l.lg/1 1 of solution). Sodium azide (0.02% w/v) was added to incubation media to inhibit bacterial growth. The trypsin solution was prepared once before the experiment and the activity measured by caseinolytic method was 2.844 U/ccm. After six days of the experiment it decreased to 1.558 U/ccm The samples after treatment in trypsin were rinsed with deionized water. 

Trypsin digestion Add enough of a 0.2 pg/pL trypsin solution to swell the gels and incubate on ice or at 4 C for 15 min. Usually 20 pL is sufficient. The trypsin solution can be prepared and ahquoted ahead of time (20 microfuge tubes of 10 pL each) and stored in the freezer until ready. 

FIGURE 3.9 On-tissue digestion process was enhanced by the heat-denaturation process and the use of detergent-supplemented trypsin solution. A. Mass spectra obtained from 10-pm mouse cerebellum sections of trypsin-digested position (see inset). B-D. Mass spectrum obtained from the tissue sections prepared with indicated treatment of denaturation and detergent. Asterisks represent the mass peaks at m/z 1198.7. E. Signal-to-noise ratio of peak at m/z 1198.7 obtained from each tissue section. Bar, 1 pm [19]. 

Open tubular capillary microreactor Trypsin Entrapment Immobilized trypsin reactor prepared by enacapsulating trypsin in hydrogel. Enzymatic activity 700 times higher than enzyme in free solution. Enzymatic activity was evaluated using a-N-benzoyl-i-arginine ethyl bradyldnin ester and two peptides (bradykinin and [Tyr8]-) [107]... 

Recovery. The principal purpose of recovery is to remove nonproteinaceous material from the enzyme preparation. Enzyme yields vary, sometimes exceeding 75%. Most industrial enzymes are secreted by a microorganism, and the first recovery step is often the removal of whole cells and other particulate matter (19) by centrifugation (20) or filtration (21). In the case of ceU-bound enzymes, the harvested cells can be used as is or dismpted by physical (eg, bead mills, high pressure homogenizer) and/or chemical (eg, solvent, detergent, lysozyme [9001 -63-2] or other lytic enzyme) techniques (22). Enzymes can be extracted from dismpted microbial cells, and ground animal (trypsin) or plant (papain) material by dilute salt solutions or aqueous two-phase systems (23). 

Trypsinize HeLa cells and prepare cells at a concentration of 50,000 cells/mL diluted in DMEM containing FBS and L-glu-tamine (see Note 8). Add 20 pL of cell solution (final concentration of 1,000 cells/well) to all plates. 

Tryptophane is not obtained in any large amount by the hydrolysis of proteins by acids and is best prepared by the action of trypsin. According to Hopkins and Cole, the protein is digested in alkaline solution by trypsin, until the solution gives a maximal coloration when tested with bromine water the solution is then acidified, boiled and... 

Because high levels of display can impair phage infection, we recommend treating the phages with 10-7 M trypsin for 30 min before measuring the titer. Note that trypsin removes the displayed protein only if a cleavable linker is used or if the protein itself is degraded by trypsin. The stock solution of trypsin (10-5 M) should be freshly prepared in 20 mM acetate buffer, pH 3.0. 

Modified trypsin is preferred for protein digestion, as it is less susceptible to autodigestion (7). The trypsin 10X stock solution is prepared by dissolving the trypsin (20 gig) in the solvent supplied with the enzyme (50 mMacetic acid 200 pL). This is stored at -70°C in aliquots (10 pL) and is stable for at least 1 yr. 

For many years a solution of crude acetone powder of bovine or porcine pancreas has been used to disaggregate tissues and to release cultured cells from their substratum. These preparations — referred to as trypsin 1 250 (based on an international standard) — contain not only trypsin but a range of enzymes including chymotrypsin and elastase which are equally important. Purified trypsin seldom is as efficient as the cruder preparations, especially on tissue disaggregation, when mucinous clumps result (Ronaldoni, 1959). 

Enzymatic gelation of partially heat-denatured whey proteins by trypsin, papain, pronase, pepsin, and a preparation of Streptomyces griseus has been studied (Sato et al., 1995). Only peptic hydrolysate did not form a gel. The strength of the gel depended on the enzyme used and increased with increasing DH. Hydrolysis of whey protein concentrate with a glutamic acid specific protease from Bacillus licheniformis at pH 8 and 8% protein concentration has been shown to produce plastein aggregates (Budtz and Nielsen, 1992). The viscosity of the solution increased dramatically during hydrolysis and reached a maximum at 6% DH. Incubation of sodium caseinate with pepsin or papain resulted in a 55-77% reduction in the apparent viscosity (Hooker et al., 1982). 

This approach was reported by Carell et al. [30] and, whilst it has often been cited with the name subtractive deconvolution, it could be also named tailored deconvolution. One of the libraries deconvoluted by this method, and targeted to trypsin inhibition, was prepared in solution adding equimolar amounts of 19 amino acids (Gin excluded) to a tetrafunctional xanthene scaffold, to produce a solution library composed of 65,341 individuals as a single pool (Figure 8.7). The extensive chemical and analytical assessment which assured the quality of the library included synthesis of six smaller model libraries (30-60 compounds), using simplified xanthene scaffolds, and their thorough MS characterization. Further details are not presented here, being out of the purpose of this review, but the conclusions about the library quality can be safely accepted. 

Sample Preparations Dissolve a sufficient amount of sample, accurately weighed, in 0.001 N hydrochloric acid to produce a solution containing about 3000 USP trypsin units in each mL. Prepare three dilutions using 0.001 A hydrochloric acid so that the final solutions will contain 12, 18, and 24 USP trypsin units in each 0.2 mL. Use these concentrations in the Procedure below. 

Pipet 0.2 mL of 0.001 N hydrochloric acid and 3.0 mL of Substrate Solution into a 1-cm cell. Place this cell in the spectrophotometer, and adjust the instrument so that the absorbance will read 0.050 at 253 nm. Pipet 0.2 mL of the Sample Preparation containing 12 USP units into another 1-cm cell. Add 3.0 mL of Substrate Solution, and place the cell in the spectrophotometer. At the same time the Substrate Solution is added, start a stopwatch, and read the absorbance at 30-s intervals for 5 min. Repeat the procedure with the Sample Preparations containing 18 and 24 USP units. Plot curves of absorbance versus time for each concentration, and use only those values that form a straight line to determine the activity of the trypsin. Discard the values on the plateau, and take the average of the results from the three concentration levels as the actual activity of the trypsin. 

Protein molecular weight standards—Suggested standards are phosphorylase (97,400 Da), bovine serum albumin (66,200 Da), ovalbumin (45,000 Da), carbonic anhydrase (31,000 Da), soybean trypsin inhibitor (21,500 Da) and lysozyme (14,400 Da) at 1 mg/ml each in a single mixture in 0.01 M Tris chloride buffer, pH 7.0 (prepared by dilution of the stock 1 M buffer above). Approximately 2 ml of this solution will be required. 

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