基本描述

产品名称	IdeZ Protease (IgG-specific, Powder)
别名	IdeZ 蛋白酶（IgG 特异性，粉末）
规格或纯度	≥95%(SDS-PAGE), 不含除IdeZ Protease之外的其它种类蛋白酶，不含DNA内切酶和外切酶，不含RNA酶，不含磷酸酯酶。
产品介绍	阿拉丁生产的IdeZ Protease (IgG-specific)，即Immunoglobulin-degrading Enzyme from Streptococcus Equi Subspecies Zooepidemicus Protease，也称免疫球蛋白特异性IdeZ蛋白酶，是由阿拉丁自主研发的PerfectProtein™技术平台表达、纯化获得的一种免疫球蛋白IgG降解酶(Immunoglubulin G-degrading enzyme)。IdeZ Protease具有极高的底物特异性，仅能识别人等特定种属和特定类型的IgG，并在抗体铰链区下方的特定位点进行酶切，使IgG水解为完整的F"2', "片段和Fc片段(参考图1)。IdeZ Protease作为工具酶主要应用于抗体类药物或抗体融合蛋白药物的结构表征分析，制备仅包含F"2片段的抗体或抗体的Fc片段，也可用于免疫沉淀后Western检测时去除重链对于目标蛋白检测的干扰等用途。图1. IdeZ Protease (IgG-specific)用于切割人IgG的效果示意图。IdeZ Protease酶切后，在非还原性和非变性条件下可以产生100kD的F"2', "和50kD的Fc；如果在变性和还原性条件下会产生2个25kD的Fd'片段，2个25kD的LC片段和2个25kD的Fc/2片段。酶切过程中或非酶切过程中都可使用"PNGase F 对抗体进行去糖基化。IdeZ Protease可有效切割人IgG1、IgG2、IgG3和IgG4，还可以切割兔、大鼠、猴、绵羊(Sheep)、人源化和嵌合的IgG以及Fc融合蛋白，如Enbrel 1]。但IdeZ Protease不能切割小鼠IgG1/IgG2b，猪、牛或山羊IgG，以及非IgG同种型免疫球蛋白，包括IgA、IgM、IgD和IgE。与IdeS Protease相比，IdeZ Protease对大鼠、小鼠IgG2a和IgG3的酶切活性显著提高。阿拉丁生产的IdeZ Protease (IgG-specific)用于切割人、兔和驴IgG的效果参考图2。图2. 阿拉丁生产的IdeZ Protease (IgG-specific, Powder) 用于切割人、兔和驴IgG的效果图。在25µl反应体系(50mM Sodium Phosphate (pH7.5 @25℃))中，加入10μg人、兔和驴IgG ，再加入2.5µl的10X Reaction Buffer以及图中指定量的本产品，然后用超纯水补至25µl，37℃孵育30分钟进行酶切。反应结束后，立即65℃孵育10分钟以终止反应。取出10μl反应产物，加入2μl SDS-PAGE蛋白上样缓冲液(6X) ，进行12%聚丙烯酰胺凝胶电泳和考马斯亮蓝染色，拍照观察结果。实验结果表明，本产品对人和兔IgG具有明显的切割效果，并且对于兔IgG有非常高的酶活性，但不能特异性识别与切割驴IgG。实际使用效果会因实验条件、实验材料等的不同而存在差异，图中效果仅供参考。阿拉丁生产的IdeZ Protease (IgG-specific)用于切割大鼠IgG的效果参考图3。图3. 阿拉丁生产的IdeZ Protease (IgG-specific, Powder) 用于切割大鼠IgG的效果图。在25µl反应体系(50mM Sodium Phosphate (pH7.5 @25℃))中，加入10μg大鼠IgG ，再加入2.5µl的10X Reaction Buffer以及图中指定量的本产品，然后用超纯水补至25µl，37℃孵育120分钟进行酶切。反应结束后，立即65℃孵育10分钟以终止反应。取出10μl反应产物，加入2μl SDS-PAGE蛋白上样缓冲液(6X) ，进行12%聚丙烯酰胺凝胶电泳和考马斯亮蓝染色，拍照观察结果。实验结果表明，本产品能识别并切割大鼠IgG，但酶切效率相对较低，需要增加使用酶量或延长反应时间才能达到充分切割的目的。实际使用效果会因实验条件、实验材料等的不同而存在差异，图中效果仅供参考。本产品具有良好的酶切活性并含有His标签，在酶切完成后可以使用镍柱很容易地从酶切反应体系中分离去除含有His标签的IdeZ Protease。分子量：约为33kDa。用途：抗体类药物或抗体融合蛋白药物的结构表征分析，酶切产生制备仅包含F", '2片段的抗体或抗体的Fc片段，也可以用于免疫沉淀后Western检测时去除重链对于目标蛋白检测的干扰等用途。来源：纯化自携带编码IdeZ Protease基因的E.coli重组菌株。酶储存溶液： Lyophilized from 50mM Sodium Phosphate, 150mM NaCl (pH6.6 @25℃)。IdeZ Protease Dilution Buffer: 50mM Sodium Phosphate, 150mM NaCl (pH6.6 @25℃)。10X Reaction Buffer: 500mM Sodium Phosphate (pH7.5 @25℃)。失活或抑制： 65℃孵育10分钟，可使IdeZ Protease失活。注意事项：避免反复冻融，建议首次使用时适当分装后冻存使用。IdeZ Protease使用时宜存放在冰盒内或冰浴上，使用完毕后宜立即放置于-20℃保存。IdeZ Protease对不同物种IgG的酶切效率存在较大差异，酶切特定物种的IgG时，建议适当摸索反应孵育时间和使用酶量。IdeZ Protease对小鼠IgG2a和IgG3的切割效率偏低，建议增加IdeZ Protease的使用量(建议5倍至10倍作为起点)或延长反应孵育时间(2小时至过夜)以实现对小鼠IgG2a和IgG3的充分酶切。IdeZ Protease在中性pH或接近中性pH的缓冲体系中活性最强，且酶切体系中盐浓度不宜过高，推荐使用本产品提供的10X Reaction Buffer进行酶切反应。在使用pH6-8范围以外的缓冲液(例如醋酸盐缓冲液)时，孵育时间或酶量需要根据实际情况进行优化。本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。为了您的安全和健康，请穿实验服并戴一次性手套操作。使用说明： 1.首次打开管盖前，建议8,000-12,000×g离心约10秒，使附着在管盖或管壁上的蛋白聚集于管底。随后取IdeZ Protease (Powder)，加入适量超纯水配制成终浓度为10U/μl的工作液，按照20μl/管分装到1.5ml的离心管中，-20℃保存，以避免反复冻融影响酶活。2.室温解冻IdeZ Protease，并参考下表在冰浴中配制反应体系(以25μl体系为例)。 Reagent Volume 10X Reaction Buffer 2.5µl Ultrapure water (16.5-x) μl Substrate (IgG) xμl (up to 10μg) IdeZ Protease (10U/μl) 1μl (10units) Total Volume 25µl 注1：建议最后添加IdeZ Protease。注2：酶切不同物种的IgG，建议适当摸索最佳孵育时间和酶浓度。对于小鼠IgG2a和IgG3的酶切，建议增加酶量(建议5倍至10倍作为起点)或延长反应孵育时间(2小时至过夜)以实现充分酶切。注3：如需减少酶量，建议使用本产品提供的IdeZ Protease Dilution Buffer对IdeZ Protease进行稀释，稀释过程中请勿使用Vortex剧烈振荡，以免蛋白变性而失活。注4：在使用pH6-8范围以外的缓冲液(例如醋酸盐缓冲液)时，孵育时间和酶量需要根据实际情况进行优化。3.用移液器轻轻吹打混匀或轻微Vortex混匀，随后低速离心以使粘附在管壁上的液体聚集于管底。4.反应条件：37℃孵育30-60分钟。5.终止反应：65℃孵育10分钟。6.将反应后的产物进行变性或非变性PAGE凝胶电泳，考马斯亮蓝染色后拍照观察并分析酶切效果。7.其它用途请自行参考IdeZ Protease的相关文献资料进行。常见问题：1.与IdeS Protease相比，IdeZ Protease的优势是什么？IdeZ Protease与IdeS Protease具有相同的特异性，两者都是在IgG铰链区域下方完全相同的单个位点进行切割。但IdeZ Protease的酶切效率高于IdeS Protease，能够更有效地切割小鼠IgG2a。因此，IdeZ Protease比IdeS Protease更通用。2.IdeZ Protease在磷酸盐缓冲液(PBS)中具有活性吗？是的，IdeZ Protease在磷酸盐缓冲液(PBS)中具有活性。IdeZ Protease能够在pH范围为6-9的缓冲体系中发挥酶切活性，但是在pH5.5以下，其活性显著降低。推荐使用本产品提供的10X Reaction Buffer进行酶切反应。3.IgG经IdeZ Protease酶切后，能否使用Protein A磁珠或Protein A Agarose纯化富集Fab和Fc片段？可以。在用IdeZ Protease切割后，可使用protein A磁珠或Protein A Agarose富集Fc和Fab片段。Fc片段会与磁珠或Agarose结合，Fab片段仍保留在上清中。注明仅和Fc片段结合的Protein G磁珠或Protein G Agarose，也可用于分离Fc和Fab片段。4.IdeZ Protease能否切割除人以外其他物种的IgGs？IdeZ Protease可有效切割人、人源化、嵌合、绵羊、兔、大鼠和猴IgG；延长孵育时间也可切割小鼠IgG2a和IgG3；还可以切割许多Fc融合蛋白以及抗体药物偶联物(ADC)。但IdeZ Protease不可用于切割山羊、牛、猪IgG，以及小鼠IgG1或IgG2b。5.IdeZ Protease能否切割除IgG以外的其它类型Ig？不能，IdeZ Protease仅能有效切割IgG，对于IgA、IgD、IgE或IgM没有切割活性。6.IdeZ Protease对IgG的特异性切割位点是什么? IdeZ Protease对人和小鼠的特异性识别与切割位点如下表所示： Subclass Hinge/CH2 sequence IdeZ Activity Human IgG1 CPPCPAPELLG^GPSVF + + + Human IgG2 CPPCPAPP_VA^GPSVF + + + Human IgG3 CPRCPAPELLG^GPSVF + + + Human IgG4 AHHAQAPEFLG^GPSVF + + + Mouse IgG1 PCICTVPEV_SSVF - Mouse IgG2a CPPCAAPNLLG^GPSVF + + + Mouse IgG2b CHKCPAPNLEGGPSVF - Mouse IgG3 GSSCPAGNILG^GPSVF + + + 7.在同一反应体系中，PNGase F能否和IdeZ Protease一起使用，以便对抗体的Fc部分进行去糖基化？可以。PNGase F可以与IdeZ Protease在同一个步骤中一起使用，实现对IgG的酶切片段化，同时去除Fc的聚糖。推荐使用阿拉丁生产的PNGase F 对抗体进行去糖基化处理。参考文献：1.Lannergård J, Guss B. FEMS Microbiol Lett. 2006. 262(2):230-5. Aladdin's IdeZ Protease (IgG-specific) is an Immunoglobulin G-degrading Enzyme recombinantly expressed in E. coli and purified using the PerfectProtein™ Platform developed by aladdin.IdeZ Protease is highly substrate specific. It recognizes only IgG of specific species and cleaves the IgG at a specific site below the hinge region into F2 and Fc fragments (Figure 1). IdeZ Protease is mainly used for structure characterization of antibodies or antibody-fusion protein drugs, preparation of antibodies containing only the F2 fragment or the Fc fragment of antibodies, and removal of heavy chains from immunoprecipitated products for Western blot analysis.Figure 1. Schematic diagram of Aladdin's IdeZ Protease (IgG-specific) for cleavage of human IgG. Under non-reducing and non-denaturing conditions, the cleavage products demonstrate 100kD F2 and 50kD Fc fragments. However, under denaturing and reducing conditions, the cleavage products demonstrate two 25kD Fd' fragments, two 25kD LC fragments and two 25kD Fc/2 fragments. The antibody can be deglycosylated using the PNGase F during or outside the enzymatic cleavage process.IdeZ Protease efficiently cleaves human IgG1, IgG2, IgG3 and IgG4, as well as rabbit, rat, monkey, sheep, humanised and chimeric IgG and Fc fusion proteins such as Enbrel [1]. However, IdeZ Protease does not cleave mouse IgG1/IgG2b, porcine, bovine and goat IgG, nor non-IgG isoforms of immunoglobulins including IgA, IgM, IgD and IgE. Compared to IdeS Protease, IdeZ Protease has significantly higher cleavage activity for rat and mouse IgG2a and IgG3.Please refer to Figure 2 for the performance of this product in cleaving human, rabbit, and donkey IgG.Figure 2. The digestion effect of Aladdin's IdeZ Protease (IgG-specific) on human, rabbit, and donkey IgG. Ten micrograms IgG of Human, Rabbit and Donkey origin in 25μl reactions, respectively, were digested according to the instructions of this product. Reaction products of 10μl were mixed with 2µl of SDS-PAGE Protein Loading Buffer (6X) , then subjected to 12% PAGE analysis, followed by gel staining with the UltraBio™ Coomassie Blue Super Fast Staining Solution . The experimental results show that this product cleaves human and rabbit IgG effectively, but not donkey IgG. This figure is for reference only, which may vary due to different experimental conditions.Please refer to Figure 3 for the performance of this product in cleaving rat IgG.Figure 3. The digestion effect of Aladdin's IdeZ Protease (IgG-specific) on rat IgG. Ten micrograms rat IgG in 25µl reactions were digested by indicated amounts of IdeZ Protease at 37℃ for 120 minutes according to the instructions of this product. Reaction products of 10μl were mixed with 2µl of SDS-PAGE Protein Loading Buffer (6X) , then subjected to 12% PAGE analysis, followed by gel staining with the UltraBio™ Coomassie Blue Super Fast Staining Solution . The experimental results show that this product can cleave rat IgG, but with lower efficiency, and thus it is necessary to increase the amount of enzyme or extend the reaction time to achieve a complete cleavage of rat IgG. This figure is for reference only, which may vary due to different experimental conditions.This product is a His tag fusion protein with good enzymatic activity, which allows for easy separation of IdeZ Protease from the reaction using a nickel column after enzymatic cleavage.Molecular weight Application： Structure characterization of antibody drugs or antibody-fusion protein drugs; preparation of antibodies containing only the F2 fragment or the Fc fragment of antibodies; removal of heavy chains from immunoprecipitated products for Western blot analysis. Source： Purified from E. coli with recombinant expression of IdeZ Protease.Enzymatic activity: IdeZ Protease (IgG-specific) is a recombinant antibody specific protease that cleaves IgG of human, rabbit, rat, sheep and monkey origin. It cleaves specifically at a single recognition site below the hinge region to yield a homogenous pool of F2 and Fc fragments. IdeZ Protease (IgG-specific) cleaves murine IgG2a more effectively than IdeS Protease. Definition of enzymatic activity unit: One unit is defined as the amount of enzyme required to cleave ＞95% of 1µg of human IgG, in 15 minutes at 37℃ in a total reaction volume of 10µl.Purity: ＞95% by SDS-PAGE analysis. No other proteases besides IdeZ Protease, no DNA endonucleases or exonucleases, no RNases, and no phosphatases.Storage of Enzyme: Lyophilized from 50mM Sodium Phosphate, 150mM NaCl (pH6.6 at 25℃).IdeZ Protease Dilution Buffer: 50mM Sodium Phosphate, 150mM NaCl (pH6.6 at 25℃).10X Reaction Buffer: 500mM Sodium Phosphate (pH7.5 at 25℃). Inactivation or inhibition： IdeZ Protease can be inactivated by incubation at 65℃ for 10 minutes. Precautions： IdeZ Protease should be kept on ice during use, and immediately stored at -20℃ after use.IdeZ Protease has different enzyme activity towards IgG from different species. The reaction time and enzyme amount should be adjusted accordingly. As the IdeZ Protease shows low efficiency in cleaving mouse IgG2a and IgG3, the enzyme amount should be increased by 5-10 times or the reaction time should be extended (e.g., 2 hours to overnight) to achieve a complete digestion of mouse IgG2a and IgG3.IdeZ Protease has the highest activity under neutral or close-to-neutral pH conditions, and the salt concentration should not be too high. We recommend using the 10X Reaction Buffer provided with this product for the digestion reaction. When using buffers outside the pH range of 6-8, such as acetate buffer, the reaction time or enzyme amount needs to be optimized accordingly.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation. Instructions for Use： 1.Before opening the tube for the first time, centrifuge at 8,000-12,000×g for approximately 10 seconds to collect powder to the bottom of the tube. Solubilize the IdeZ Protease (Powder) in an appropriate amount of ultra-pure water to obtain a working solution of 10U/μl. Aliquot the reconstituted IdeZ Protease into microcentrifuge tubes at a volume of 20μl per tube, and store at -20℃ to avoid repeated freeze-thaw.2.Set up the following reaction in a new microcentrifuge tube on ice. Reagent Volume 10X Reaction Buffer 2.5µl Ultrapure water (16.5-x) μl Substrate (IgG) xμl (up to 10μg) IdeZ Protease (10U/μl) 1μl (10 units) Total Volume 25µl Note 1: We recommend adding the IdeZ Protease at last.Note 2: When cleaving IgG from different species, the optimal reaction time and enzyme amount should be determined accordingly. For the cleavage of mouse IgG2a and IgG3, increase the enzyme amount by 5-10 times or extend the reaction time (e.g., 2 hours to overnight) to achieve full digestion.Note 3: If it is necessary to reduce the amount of enzyme, we recommend diluting IdeZ Protease with the IdeZ Protease Dilution Buffer provided with this product. Do not vortex to avoid protein denaturation.Note 4: When using a buffer outside the pH range of 6-8, such as acetate buffer, the incubation time and enzyme amount should be optimized.3.Gently mix the reaction by pipetting or vortex. Then centrifuge at low speed to collect the liquid at the bottom of the tube.4.Incubate at 37℃ for 30-60 minutes.5.Terminate the reaction by incubating at 65℃ for 10 minutes.6.Perform denaturing or non-denaturing PAGE analysis and then stain with Coomassie Brilliant Blue to analyze the digestion result.7.For other applications, please refer to relevant literature.FAQ:1.What are the advantages of IdeZ Protease compared to IdeS Protease?IdeZ Protease and IdeS Protease have the same specificity in cleaving the same single site below the hinge region of IgG. However, IdeZ Protease is more efficient in cleaving mouse IgG2a than IdeS Protease and is therefore more versatile than IdeS Protease.2.Does IdeZ Protease have activity in phosphate-buffered saline (PBS)?Yes, IdeZ Protease has activity in phosphate-buffered saline (PBS). IdeZ Protease can exert enzymatic activity in buffers with pH range of 6-9, but its activity is significantly reduced below pH 5.5. We recommend using the 10X Reaction Buffer provided with this product for the digestion reaction. 3.Can Protein A magnetic beads or Protein A agarose be used to purify and enrich Fab and Fc fragments of IgG after cleavage by IdeZ Protease?Yes. The Fc fragment will bind to the magnetic beads or agarose, while the Fab fragment will remain in the supernatant. Protein G magnetic beads or Protein G agarose gels that only bind to the Fc fragment can also be used to separate Fc and Fab fragments.4.Can IdeZ Protease cleave IgGs from species other than humans?IdeZ Protease efficiently cleaves human, humanised, chimeric, sheep, rabbit, rat and monkey IgG. It also cleaves mouse IgG2a and IgG3 with extended reaction time, Fc fusion proteins, and antibody drug couples (ADCs) as well. However, IdeZ Protease cannot cleave goat, bovine and porcine IgG, mouse IgG1 or IgG2b.5.Does IdeZ Protease cleave other types of immunoglobins besides IgG?No, IdeZ Protease only effectively cleaves IgG and does not cleave other types of immunoglobulins such as IgA, IgD, IgE and IgM.6.What is the specific cleavage site for IdeZ Protease?The specific recognition and cleavage sites for IdeZ Protease are as follows: Subclass Hinge/CH2 sequence IdeZ Activity Human IgG1 CPPCPAPELLG^GPSVF + + + Human IgG2 CPPCPAPP_VA^GPSVF + + + Human IgG3 CPRCPAPELLG^GPSVF + + + Human IgG4 AHHAQAPEFLG^GPSVF + + + Mouse IgG1 PCICTVPEV_SSVF - Mouse IgG2a CPPCAAPNLLG^GPSVF + + + Mouse IgG2b CHKCPAPNLEGGPSVF - Mouse IgG3 GSSCPAGNILG^GPSVF + + + 7.Can PNGase F be used together with IdeZ Protease in the same reaction to deglycosylate the Fc portion of the antibody?Yes, PNGase F can be used together with IdeZ Protease in the same reaction to achieve the cleavage of IgG and the deglycosylation of Fc fragments simultaneously. ’s PNGase F can be used for the deglycosylation of antibodies.References:1.Lannergård J, Guss B. FEMS Microbiol Lett. 2006. 262(2):230-5.

产品规格参数

浓度	不含除IdeZ Protease之外的其它种类蛋白酶，不含DNA内切酶和外切酶，不含RNA酶，不含磷酸酯酶。
储存温度	-20°C储存,避免反复冻融
运输条件	超低温冰袋运输
稳定性与储存	-20℃保存，两年有效。避免反复冻融，建议首次使用时适当分装后冻存使用。
单位定义	One unit is defined as the amount of enzyme required to cleave ＞95% of 1μg of human IgG, in 15 minutes at 37℃ in a total reaction volume of 10μl.

安全和危险性（GHS）

SDS英文版

质量标准

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质检证书（CoA,COO,BSE/TSE 和分析图谱)

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货号 (SKU)	包装规格	是否现货
I747010-1KU	1KU	期货
I747010-5KU	5KU	期货
I747010-20KU	20KU	期货

IdeZ Protease (IgG-specific, Powder)

库存信息

库存信息

库存信息

基本描述

产品规格参数

安全和危险性（GHS）

质量标准

质检证书（CoA,COO,BSE/TSE 和分析图谱)

溶液计算器

摩尔浓度计算器

稀释计算器

复溶计算器