| Product Description | 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.Related Products：Cat. 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