- -related" Topic
- Parsing protease activity assay Focused protease research new progress
1g solid enzyme powder (or 1mL liquid enzyme), at a certain temperature and pH, 1min hydrolytic tyrosine produces 1ug tyrosine as an enzyme vitality unit, expressed in (u/mL).
2 forin method (first method)
2, 1 principle
. Under the condition of temperature and PH, hydrolyzed substrate, producing amino acids (e.g., tyrosine, tryptophan, etc.), under alkaline conditions, the forintin > (Folin) reduced, produced tantalum and tungsten blue, using the luminosity method to determine its enzyme vitality.
2, 2 reagents and solutions
2, 2, 1 forling reagent preparation>
adds sodium tungstenate (Na2Wo4.2H2O) 100g, sodium tantalum (Na2MoO4.2H2) to the 2000mL reflow unit O) 25g, water 700mL, 85% phosphate 50mL, thick hydrochloric acid 100mL, water and fire boiling reflow 10h, remove the reflow cooler, add lithium sulfate (Li2SO4) 5 in the ventilation cabinet 0g, water 50mL and a few drops of thick bromine water (99%), then slightly boil 15min, to remove excess bromine (after cold people have green need to add brominated water, and then boil to remove the amount of bromine), cooling, see water dissolved to 1000ml. Mix well, < a href"" > filter . The reagents should be golden brown and stored in brown bottles.
Use solution: a forin reagent mixed with two servings of water, shake well.
2, 2, 2 sodium carbonate solution c (Na2CO3) s0.4mol/L
said no water sodium carbonate (Na2CO3) 42.4g, dissolved with water and fixed to 1000 mL.
2, 2, 3 tetluchloroacetic acid c (CCl3. COOH) s 0.4mol/L
says take 65.4g of triclosan, dissolve with water and set the capacity to 1000 mL.
2, 2, 4 sodium hydroxide solution c (NaOH) s 0.5mol/L
is made by GB601.
2, 2, 5 hydrochloric acid solution c (HCI)=1 mol/L and 0.1 mol/L
2, 2, 6 buffer solution
a, phosphoric acid buffer (PH=7.5) suitable for neutral proteases
is called 6.02g of na2HPO4.12H2O and 0.5g of sodium phosphate (NaH2PO4.12H2O), dissolved with water and settled to 1000mL.
b, lactic acid buffer (PH=3.0) is suitable for acid protease
A liquid is called lactic acid (80% to 90%) 10.6g, dissolved with water and fixed to 1000 mL.
B liquid is called sodium lactic acid (70%) 16g, dissolved with water and fixed to 1000 mL.
use solution to extract 8 mL of acetylene, add b mixture 1 mL, mix well, dilute twice, i.e. 0.05moi/L lactic acid buffer solution.
c, boric acid buffer solution (PH=10.5) suitable for alkaline protease
A liquid called sodium borate (borax) 19.08g, water dissolved and fixed to 1000 mL.
B liquid is called sodium hydroxide 4.0g, water dissolved and fixed to 1000 mL.
use solution to extract 500 mL of acetylene, 400 mL of b liquid mixed well, diluted with water to 1000mL.< p style is "text-align: left;" > of the above buffer solutions must be corrected with a PH meter.
2, 2, 7 10g/L casein 3) solution
called casein 1.000g, accurate to 0.001g, with a small amount of 0.5mol/L sodium hydroxide solution (if acidic protein). Lactic acid 2 to 3 drops) moistened, add a moderate amount of various suitable PH buffer solution about 80 mL, in the boiling water bath< >p style< text-align: > left; This solution is stored in the refrigerator and is valid for three days.
Note: 3) tyrosine using Shanghai chemical reagents procurement supply station distribution reagents.
2, 2, 8 100ug/mL L-tyrosine 4) standard solution
a, called pre-105 degrees C dry to constant L-tyrosine 0.1000g, accurate to 0.0002g, with 1mol/L hydrochloric acid 60 mL dissolved to 100 mL, that is, 1mg/mL tyrosine standard solution.
Note:4)L-Tyrosine uses Shanghai Changjiang " pharmaceutical plant products.
b, absorb 1mg/mL tyrosine standard solution 10.00 mL, with 0.1mol/L hydrochloric acid to 100 mL, i.e. get 100ug/mL L-Tyrosine standard solution.
2, 3 instruments and equipment
< text-align:left;"> 2, 3, 1 < a href"">" water bath 40±0.2 degrees C.< p-style" text-align: left;"> 2, 3, 2 diclight meters should comply with GB9721.
2, 4 analysis steps
< text-align:left;">2, 4, 1 standard curve drawing
a, L-tyrosine standard solution
< left;"> >< Table 3