protease-negative mutant是什么意思

英语翻译
请不用机译
Measurement of protease activity by azocasein assay
The assays were conducted as described previously (Scholze and Tannich 1994) with some minor modifications.Briefly,parasitic lysates were preincubated with 2 mM dithriothreitol (DTT) (Sigma) at 37°C for 10 min to activate protease activity.100 μl of 5 mg/ml azocasein (Sigma) solution was prepared in PBS (pH 7.4) and incubated with 100 μl of parasitic lysate for 1 h at 37°C.Reaction was stopped by adding 300 μl of 10% trichloroacetic acid and samples were incubated on ice for 30 min.Undigested azocasein was removed by centrifugation (5,000×g,5 min) and the resulting supernatant was transferred to a clean tube containing 500 μl of NaOH (525 mM).Absorbance was measured at 442 nM on a spectrophotometer (Tecan Magellan).For controls,lysates were boiled at 90°C for 15 min to inactivate proteases and 100 μl trypsin (2.5 mg/ml) was used as a positive control.
Analysis of proteases by nondenaturing gelatin-SDS-PAGE
This SDS-PAGE method requires the inclusion of a protein,usually gelatin,in an acrylamide gel.Gelatin is digested by proteases after electrophoresis and clear bands are seen after staining the gel with Coomassie Brilliant blue.Cell lysate (40 μg) of B isolate was analyzed by gelatin-SDS-PAGE for protease activity (Lockwood et al.1987) using the SDS-discontinuous buffer system of Laemmli (1970).The acrylamide concentration of resolving and stacking gel was 12.5 and 5%,respectively.Briefly,0.2% (w/v) gelatin (Sigma) was copolymerized into the resolving gel.Samples were not boiled so that enzymatic activity of the proteases upon renaturation would not be lost.After electrophoresis at a constant current of 30 mA/gel for 1.5 h,gels were immersed with continuous shaking for 1 h in 2.5% (v/v) Triton X-100 to remove the SDS and to allow the proteases to become active.The proteinases bands were developed by immersing the gels in incubation buffer containing 1 mM DTT for 3 h at 37°C.The bands were visualized by staining in 0.12% (w/v) Coomassie Brilliant blue R-250 for 30 min.Cell lysate was also analyzed by 12.5% gelatin-containing native gel (without SDS) to determine the number of proteases present and to investigate whether the proteases were single/multiple subunits.
Inhibition of proteases
To determine the type of proteases that are present in Blastocystis,effects of inhibitors on the activity of Blastocystis proteases were studied by incubating Triton-treated gels in incubation buffer with 1 mM DTT containing one of the following inhibitors:iodoacetamide (IA),EDTA,N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK),8-hydroxyquinoline,phenylmethylsulfonyl fluoride (PMSF) (all 1 mM); pepstatin A,leupeptin or antipain (all 100 μg/ml).All inhibitors were purchased from Sigma except pepstatin A (CHEMICON),IA (MP Biomedicals,LLC) and PMSF (BDH).Gels were stained with Coomassie Brilliant blue.
For azocasein assays,similar concentrations of protease inhibitors (IA,TLCK,pepstatin A,EDTA) were added during a 1-h incubation of parasitic lysate with azocasein.
Determination of pH optimum
The pH dependence of Blastocystis protease activity was investigated by incubating Triton X-100 treated gels in buffers of different pH ranging from 3.0 to 8.5.The following buffers were used:0.1 M glycine/HCl (pH 3.0),0.1 M sodium acetate/acetic acid (pH 4.0 and 5.5),0.1 M sodium phosphate (pH 7.0 and 7.6),and 0.1 M Tris–HCl (pH 8.5).All incubation buffers contained 1 mM DTT and gels were stained with Coomassie Brilliant blue.For azocasein assay,a 1-h incubation with parasitic lysate was performed in buffers of different pH containing 1 mM DTT.
本人仅在此先谢过上回黎明晴空所做的精彩回答!

生物化学翻译求助11
FIG. 2.
HAX-1 can be cleaved by Omi protease in vitro. In vitro translated 35S-labeled HAX-1 protein was incubated at 37 °C without (lane 1) or with 500 ng of recombinant His-Omi134–458 for 2 h (lane 2) and6h(lane 3). The reactions were resolved on SDS-PAGE, and the gel was transferred on a PDVF membrane and exposed on X-OMAT-AR film.



HAX-1 Protein Level Decreases during Cell Death—To investigate whether the level of HAX-1 protein is regulated in mammalian cells during cell death, we treated HK-2 cells with various concentrations of cisplatin or H2O2. Fig. 3 shows the protein level of HAX-1 proportionally decreases as the concentration of cisplatin is raised from 30 to 70 µM. HAX-1 protein also decreased in cells treated with increasing concentration of H2O2.

酸性蛋白酶分子式
酸性蛋白酶（Acid Protease） 目前知道它的HS编码是35079090
它的分子式是什么?CAS编码是多少啊?

不可逆的serine protease抑制剂AEBSF HCl的生物活性数据?
详情：www.***.cn/products/aebsf-hcl.html

英语翻译
Figures 2A~2C shows the activities of protease,lipase,
and amylase of crude enzyme extracts of squid viscera.
Among the three classes of digestive enzymes,the activity of
lipase was highest.The highest activities of protease,lipase,
and amylase were found in n-hexane treated squid viscera
samples comparing to SCO2 treated.The digestive enzyme
activities of squid viscera samples might be lost due to the treatment by SCO2.The loss of enzyme activity after SCO2
treatment has been argued prior research that attributed to
the interactions between CO2 and the enzyme.This means
CO2 may form covalent complexes with free amino groups
on the surface of the enzyme [20,21].