Trypsin stock

Bring each sample to 200 /jlL with 0.5 M ammonium bicarbonate and one unit of TPCK-treated trypsin. The TPCK-treated trypsin stock is prepared in 0.5 M ammonium bicarbonate and can be frozen in single use aliquots at — 20°C. 

Digest mix for one calf brain homogenate is freshly prepared by mixing 2 ml of Collagenase stock, 2 ml Trypsin stock, and 1ml DNAse stock in 15 ml of DMEMh- S. 

Prepare a trypsin stock solution by dissolving 20 mg of the enzyme in 1 mM hydrochloric acid aqueous solution to a 1-mg/ml final concentration. 

Trypsin solution Trypsin is used to facilitate blastomere separation. To make 10 ml of stock solution, dissolve 2.5 g of trypsin into 8 ml Ca -free PBS and complete to 10 ml filter and divided into 310- tl aliquots store frozen. To make 3 ml of working solution (0.25% trypsin), add 300 pi of trypsin stock to 2.7 ml Ca +-free PBS. 

By using a PI000 pipette, transfer the pieces of olfactory mucosa into a 15 mL conical tube containing 800 pL of trypsin stock solution (0.25 %) and 1.2 mL of HBSS to make a solution containing 0.1 % trypsin. Incubate tubes in a 37 °C warm bath for 45 min. Stop trypsinization by adding 5 mL DMEM/FBS plus... 

Tryptic in-gel digestion solution 10 ng/pL trypsin in 25 mM NH4HCO3. Add 25 pL of trypsin stock to 975 pL of 25 mM NH4HCO3. Prepare freshly before use. 

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. 

Cell Culture. KB cells were maintained in a humidified atmosphere of 5% carbon dioxide - 95% air at 37°C in the presence of modified Eagle s medium containing calf serum (10%), penicillin (100 jug/ml) and streptomycin (100 units/ml). Cells were routinely subcultured with 0.25% trypsin and stocks were discarded after twenty passages. All drugs were administered with fresh media 24 hours after subculture in the following concentrations TPA, 1.6 uM RA, 1.6 juM butyric acid, 2mM. Drug treatments were for 20-24 hours. Cells were harvested for enzyme assays with phosphate-buffered saline containing 0.05% EDTA and stored at -20°C in 0.32 M sucrose. 

Take an aliquot of trypsin (10 pL of 10X stock solution) from the freezer, add aqueous buffer (90 pL) and mix. 

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. 

Sterility checks should be made on all batches of media and trypsin before these are used. As some tests take several weeks to complete, it is essential that adequate stocks are maintained. The use of untested medium is a sure way to introduce contamination into all cell lines in use in a laboratory. Similarly, cell cultures should be routinely checked for contamination. Such checks will involve growth tests on the medium in which the cells have been growing as well as tests on the cells themselves. 

ES cells for stock If the ES cells are not used for electroporation, they can be frozen. Harvest the cells by trypsinization as described in step 3 of Subheading 3.2.2., count the cells and resuspend to 2 x 106 cells/mL, dispense 0.5 mL of cells to a freezing vial with 0.5 mL 2X ES freezing media. Place the freezing vials... 

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. 

The growth rate of mammalian cells in the presence of Cell-Tak adhesive was assayed to evaluate any potential adverse effects caused by this protein. Bovine corneal endothelial cell (BCE) stocks were grown to confluency in MEM plus 15% calf serum, trypsinized, and washed several times by centrifugation on MEM. Suspensions (5 x 104 cells/mL) were seeded onto untreated 35-mm dishes (control) and dishes with Cell-Tak protein (5 fig/cm2) in MEM with 15% calf serum. At various time points during the incubation at 37 °C with 5% CO2, triplicate plates were removed. The attached cells were then trypsinized from the surface, washed, and counted in a hemacytometer. 

Mouse fibroblast cells (L-929), trypsinized from stock plates, were suspended in complete Eagle s Medium at a density of 106 cells per mL. For the assay, liquid extracts (1-mL final volume) were mixed with 1-mL double strength MEM, to... 

A stock solution of rabbit Cytochrome c was made up in water at a concentration of 4.3 mg/ml. The concentration of the stock solution was determined by quantitative amino acid analysis of a 5pJ aliquot that was hydrolyzed and analyzed as previously described (4). The stock solution was diluted 5-fold with 0. IM ammonium bicarbonate or 0. IM sodium borate (both at pH 8.2) and TPCK-Trypsin in lOmM HCl was added to give a 1 20 enzyme protein solution (w/w). Digestion was carried out at 37°C for 24 hours after which the enzymatic activity was terminated by heating at 100°C for 5 min. 

A stock solution of trypsin (Sigma T-8642 or equivalent) is first standardized using the substrate p-nitrophenol-p -guanidobenzoate HCl (Sigma N-8010). By the initial burst principle (5) one can titrate the active site of trypsin and c culate the uM amount of active trypsin introduced into the rSLPI inhibition assay. By knowing the molecular weight of the trypsin (source dependent) the uM amount of rSLPI present can be deduced from the 1 1 stoichiometry of the reaction. 

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. 

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 (0.1%) Dissolve 0.5 g trypsin powder (Gibco, cat no. 0153-61-1) in 500mL saline/EDTA solution. Adjust the pH to 7.6, sterilize through a 0.22-pm filter, and store at -20°C. This constitutes a 5% stock, which needs to be diluted to 0.1% on defrosting. 

Fig. IB. The effect of SAB on the repair kinetics of potentially lethal damage in normal human fibroblasts, Ewing s sarcoma, and human lung adenocarcinoma. No inhibitor (open circles) 8SAB (closed circles). Data points are means 1 standard deviation. Both normal fibroblast and human tumor cell cultures were grown and maintained in Eagle s MEM supplemented with 10% (VA ) fetal bovine serum, streptomycin and penicillin, at S7°C in a humidified chamber of 95% air and 5% CO2. Stock cultures in exponential growth were detached by trypsinization and appropriate cell numbers plated in 25 cm flasks and incubated to permit cell attachment. Inhibitors of poly(ADP-ribose) synthetase (SAB or BZ) were added to the appropriate cultures 2 hr prior to irradiation in air at room temperature. Following irradiation, the cells were exposed to the inhibitors for 24 hr, washed, and replenished with fresh medium. Refeeding was performed every S days and after 10-14 days, cells were fixed and the colonies stained with Giemsa. Only colonies of 50 cells or more were scored as survivors. All experiments were performed in triplicate and the survival data expressed as the mean S.E. of three separate survival experiments.

Trypsin/EDTA To make 1 liter, add 8.0 g NaCl, 0.40 g KCl, 0.10 g Na2HP04 2H20, 1.0 g glucose, 3.0 g Trizma base, 0.01 g phenol red, and 2.50 g trypsin (Difco 1 250) to distilled water, adjust pH to 7.6, and bring to a total volume of 1 liter. Filter sterilize and store in aliquots at — 20°C. This stock is diluted 1 4 in saline/EDTA for use as 0.05% trypsin/EDTA solution. Store at... 

Trypsin Dilute stock trypsin solution (2.5%) 10 times with sterile HESS. Filter-sterilize (0.20- m filter) and store in aliquots at —20°C. 

Enzyme stock preparations To make 1 ml trypsin, chymotrypsin, or endoproteinase Glu C stock solution, dissolve 1 mg enzyme in 1 ml UHQ water. Distribute the solution in 50-//1 portions in 0.5-ml Eppendorf tubes, lyophilize in a vacuum centrifuge, and store at —20°C. To make 0.2 ml endoproteinase Asp-N stock solution, dissolve 10 //g enzyme in 200 //I UHQ water. Distribute in 10-/xl portions, lyophilize, and store as above. To make 100 //I carboxypeptidase Y, A, or M stock solution, dissolve 100 //g enzyme in 100 //I UHQ water. Distribute in 10-(A portions, lyophilize, and store as above. 

Suspend the dried protein pellet in 50 /rl 50 mM NH4HCO3, pH 8.0-8.3. Add 10 /xg (10 /xl of a 1 mg/ml stock) TPCK-treated trypsin (cleaves on the C-terminal side of arginine and lysine) or chymotrypsin (cleaves on the C-terminal side of phenylalanine, tyrosine, and tryptophan). 

Proteolytic enzymes 1 mg/ml stock solutions of TPCK-trypsin and chymotrypsin as described in Section C. 

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