标签归档：protease

PDQ Protease Assay™ PDQ蛋白酶分析™

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PDQ Protease Assay™

PDQ蛋白酶分析™

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储存条件：2-10℃
纯度：–
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Factor Xa Protease |NEB酶试剂 New England Biolabs

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上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

Factor Xa cleaves after the arginine residue in its preferred cleavage site Ile-(Glu or Asp)-Gly-Arg. It will sometimes cleave at other basic residues, depending on the conformation of the protein substrate (1,2,3). The most common secondary site, among those that have been sequenced, is Gly-Arg. There seems to be a correlation between proteins that are unstable in E.coli and those that are cleaved by Factor Xa at secondary sites; this may indicate that these proteins are in a partially unfolded state (Walker, I., Riggs, P., unpublished observations). Factor Xa will not cleave a site followed by proline or arginine.

产品来源

Factor Xa Protease is purified from bovine plasma and activated by treatment with the activating enzyme from Russell’s viper venom.

产品类别:
Proteases Products

应用:
Fusion Protein Cleavage,
Target Protein Insolubility ,
Protein Purification,

Protein Digestion,

Protein Expression

  • 产品组分信息

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • P8010V     -20    
        Factor Xa Protease P8010VVIAL -20 1 x 0.025 ml 1 mg/ml
    • P8010S     -20    
        Factor Xa Protease P8010SVIAL -20 1 x 0.05 ml 1 mg/ml
    • P8010L     -20    
        Factor Xa Protease P8010LVIAL -20 1 x 0.25 ml 1 mg/ml

  • 特性和用法

    单位定义

    1 μg of Factor Xa will cleave 50 μg of MBP fusion protein test substrate, MBP-ΔSal to 95% completion in a total reaction volume of 50 μl in 6 hours or less at 23°C in 20 mM Tris-HCl (pH 8.0 @ 25°C) with 100 mM NaCl and 2 mM CaCl2.

    使用浓度

    1 mg/ml

    去除

    Factor Xa will bind specifically to benzamidine-agarose.

    分子量

    理论上的: 43 kDa

    停用

    Dansyl-glu-gly-arg-chloromethyl ketone (CALBIOCHEM, #251700) will irreversibly inactivate Factor Xa by covalent attachment at the active site. In a reaction containing 20 μg/ml Factor Xa, 2 μM dansyl-glu-gly-arg-chloromethyl ketone will inactivate >95% of the Factor Xa in 1 minute at room temperature.

  • 注意事项

    1. The test substrate MBP-ΔSal is maltose-binding protein fused to a truncated form of paramyosin, with the amino acids Ile-Glu-Gly-Arg at the fusion joint. Greater than 95% of the fusion protein is cleaved in 6 hours or less.

  • 参考文献

    1. Nagai, K. et al. (1985). Proc. Natl. Acad. Sci. USA. 82,
    2. Quinlan, R.A. et al (1989). Cell Sci. 82, 7252-7255.
    3. Eaton, D. et al (1986). Biochemistry. 25, 505-512.

操作说明、说明书 & 用法

  • 操作说明

    1. Cleavage of the Fusion Protein Generated Using The pMAL Protein Fusion and Purification System (E8200)

工具 & 资源

  • 选择指南

    • Protease Selection Chart
    • Substrate-based Ligase Selection Chart

IdeZ Protease (IgG-specific) |NEB酶试剂 New England Biolabs

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上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

IdeZ Protease (IgG-specific) |
 
IdeZ Protease (IgG-specific) is a recombinant antibody specific protease that recognizes all human, sheep, monkey, and rabbit IgG subclasses, specifically cleaving at a single recognition site below the hinge region, yielding a homogenous pool of F(ab´)2 and Fc fragments. IdeZ Protease more effectively cleaves murine IgG2a than IdeS.

IdeZ Protease (IgG-specific) |
Digestion of lgG with IdeZ Protease (lgG-specific), followed by denaturation.

产品来源

Cloned from Streptococcus equi subspecies zooepidemicus and expressed in E. coli. 

产品类别:
Proteases Products,
Proteome Analysis Products

应用:
Proteomics

  • 产品组分信息

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • P0770S     -20    
        IdeZ Protease (IgG-specific) P0770SVIAL -20 1 x 0.05 ml 80,000 units/ml
        GlycoBuffer 2 B3704SVIAL -20 1 x 1 ml 10 X

  • 特性和用法

    单位定义

    One unit is defined as the amount of enzyme required to cleave > 95% of 1 µg of human IgG, in 15 minutes at 37°C in a total reaction volume of 10 µl.

    反应条件

    1X GlycoBuffer 2
    Incubate at 37°C

    1X GlycoBuffer 2
    50 mM Sodium Phosphate
    (pH 7.5 @ 25°C)

    贮存溶液

    20 mM Tris-HCl
    50 mM NaCl
    1 mM EDTA
    pH 7.5 @ 25°C

    热失活

    65°C for 10 minutes

    分子量

    理论上的: 35578 daltons

    单位活性检测条件

    Two fold dilutions of IdeZ Protease (IgG-specific) are incubated with 1 μg of human IgG and 1X GlycoBuffer 2 in a 10 µl reaction. The reaction mix is incubated for 15 minutes at 37°C. Separation of reaction products are visualized by SDS-PAGE.

    存储注意事项

    • Avoid repeated freeze/thaw cycles.

  • 相关产品

    相关产品

    • Rapid 快速 PNGase F
    • Endo S
    • Trypsin-ultra™, Mass Spectrometry Grade
    • p6043-rapid-pngase-f-antibody-standard
    • p0711-rapid-pngase-f-non-reducing-format

  • 注意事项

    1. Store at -20°C for up to two years.
    2. IdeZ Protease efficiently cleaves human, humanized, chimeric, sheep,rabbit and monkey IgG as well as mouse IgG2a and IgG3. IdeZ Proteasewill also cleave Fc-fusion proteins, such as Enbrel.
    3. IdeZ Protease does not cleave mouse IgG1 or IgG2b, rat, porcine, bovineor goat IgG. It also does not cleave non-IgG isotypes including IgA,IgM, IgD and IgE.

操作说明、说明书 & 用法

  • 操作说明

    1. Reaction Conditions for IdeZ Protease (IgG-specific) (P0770)
    2. Reaction conditions for Simultaneous Digestion of IgG with IdeZ Protease (IgG-specific) and PNGase F (fragmentation and deglycosylation) (P0770)

FAQs & 问题解决指南

  • FAQs

    1. What is the advantage of IdeZ over IdeS?
    2. Is IdeZ Protease active in Phosphate Buffered Saline (PBS)?
    3. Can protein A magnetic beads (NEB# S1425S) be used to create Fc and Fab fragment pools after cleavage with IdeZ Protease?
    4. Can IdeZ Protease cleave IgGs other than human?
    5. Can IdeZ Protease cleave Ig isotypes other than IgG?
    6. Can PNGase F be used together with IdeZ Protease under native conditions to deglycosylate the Fc portion of an antibody?
    7. What is the cleavage site for IdeZ Protease (IgG-specific)?

TEV Protease |NEB酶试剂 New England Biolabs

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上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

TEV Protease |

TEV Protease is a highly specific cysteine protease. The TEV Protease recognition sequence with the highest catalytic efficiency is ENLYFQ ▼S; however, the amino acid in the P1’ position can also be G, A, M, C, or H (1). It is often used for the removal of affinity purification tags such as maltose-binding protein (MBP) or poly-histidine from fusion proteins. TEV Protease has a 7xHis-tag for easy removal from a reaction using nickel affinity resins and has been engineered to improve thermal stability and decrease autolysis. 

产品来源

Cloned from Tobacco Etch Virus and expressed in E. coli.

产品类别:
NEBExpress MBP Fusion and Purification System,
Bacterial E. coli Protein Expression Products,
Nickel Purification (His-tag),

Proteases Products,
Protein Purification Products,

Protein Expression Products

应用:
Fusion Protein Cleavage,
Target Protein Insolubility ,
Protein Purification,

Protein Digestion,

Protein Expression

  • 产品组分信息

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • P8112S     -20    
        TEV Protease P8112SVIAL -20 1 x 0.1 ml 10,000 units/ml
        TEV Protease Reaction Buffer B8035SVIAL -20 1 x 1 ml 10 X

  • 特性和用法

    单位定义

    1 unit of TEV Protease will cleave 2 µg of MBP-fusion protein, MBP5-TEV-paramyosin ΔSal, to 95% completion in a total reaction volume of 10 µl in 1 hour at 30°C in 50 mM Tris-HCl (pH 7.5 @ 25°C) with 0.5 mM EDTA and 1 mM DTT.

    反应条件

    1X TEV Protease Reaction Buffer
    Incubate at 30°C

    1X TEV Protease Reaction Buffer
    50 mM Tris-HCl
    0.5 mM EDTA
    1 mM DTT
    (pH 7.5 @ 25°C)

    贮存溶液

    50 mM Tris-HCl
    250 mM NaCl
    1 mM TCEP
    1 mM EDTA
    50% Glycerol
    pH 7.5 @ 25°C

    热失活

    65°C for 10 minutes

    分子量

    实际: 28 kDa

    单位活性检测条件

    Two fold dilutions of TEV Protease are incubated with 2 μg MBP5-TEV-paramyosin ΔSal and 1X TEV Protease Reaction Buffer in a 10 µl reaction. The reaction mix is incubated at 30°C for 1 hour. Separation of reaction products are visualized by SDS-PAGE.

  • 优势和特性

    Features

    • Removal of affinity purification tags such as maltose-binding protein (MBP) or poly-histidine from fusion proteins
    • Engineered to improve thermal stability and decrease autolysis
    • High substrate specificity with no non-specific proteolysis 
     

  • 注意事项

    1. The TEV Protease recognition sequence with the highest catalytic efficiency is ENLYFQ ▼S; however, the amino acid in the P1’ position (S in the recognition sequence) can also be G, A, M, C, or H (1).
    2. If the fusion protein sample contains > 2 M urea, > 0.5 M Guanidine hydrochloride, > 50 mM imidazole, pH values below 6 or above 9, or cysteine protease inhibitors then it will be necessary to dialyze the fusion protein before TEV Protease cleavage.
    3. TEV protease can be used at high concentrations without the occurrence of non-specific proteolysis.
    4. Reactions may be scaled-up linearly to accommodate larger reaction volumes.

  • 参考文献

    1. Kapust, R.B. et al. (2002). Biochem. and Biophysical Research Comm.. 294, 949-955.

操作说明、说明书 & 用法

  • 操作说明

    1. Typical Reaction Conditions for TEV Protease (NEB #P8112)
    2. His-tag removal from protein using TEV Protease

工具 & 资源

  • 选择指南

    • Protease Selection Chart

FAQs & 问题解决指南

  • FAQs

    1. Can TEV Protease recognize and cleave a sequence other than Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser)?
    2. Is it necessary to dialyze the sample prior to cleavage with TEV Protease?
    3. Can TEV Protease be used at lower temperatures?
    4. Is TEV Protease compatible with protease inhibitors?
    5. Is TEV Protease compatible with different reaction buffers?
    6. Can TEV Protease be removed from the reaction after cleavage?

  • 实验技巧

    • The optimal pH range is 6.0 – 8.0
    • Optimal activity achieved in ≤0.2M NaCl; however, the enzyme retains some activity in up to 2M NaCl
    Cleavage at lower temperatures, such as 4ºC, requires overnight incubation

    Inhibition occurs in the presence of ≥ 5 mM Zn2+, ≥ 1 mM Cu2+ and ≥ 10 mM Co2+

    • Compatible with 10mM MgSO4, MnCl2 and CaCl2 and up to 100mM EDTA
    • Compatible with the following protease inhibitors: aprotinin, benzamidine, leupeptin, pepstatin, PMSF
    • Can be used at high concentrations with no non-specific proteolysis occurring
    • Some substrates may require extended incubation (up to three days at either 4°C or 30°C)
    • If cleavage is not complete, add more TEV protease after 24 hours and continue incubation