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Pierce™ Mouse IgG1 Fab and F(ab')2 Preparation Kit Thermo Scientific™

The Thermo Scientific Pierce Mouse IgG1 Fab and F(ab')2 Preparation Kit uses immobilized ficin protease to efficiently digest mouse IgG1 into Fab or F(ab')2 fragments, depending on the chosen reaction conditions.

Features of the Mouse IgG1 Fab and F(ab')2 Preparation Kit:

High capacity—kit provides for fragmenting 0.25 to 4 mg IgG.
Complete—kit includes all reagents needed to prepare and purify antibody fragments
Enzyme-free digestion products—Immobilized Ficin (beaded agarose resin) provides for control of the digestion reaction and complete removal of resulting antibody fragments from the proteolytic enzyme
Optimized for mouse IgG1—the kit procedure is optimized for mouse IgG1; the only effective method for this IgG species and subclass (see

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Pierce™ Mouse IgG1 Fab and F(ab')2 Micro Preparation Kit
Pierce™ F(ab')2 Preparation Kit
Pierce™ Fab Preparation Kit

Pierce™ Fab Preparation Kit Thermo Scientific™

Thermo Scientific Pierce Fab Preparation Kit uses immobilized papain protease to digest human or mouse IgG antibodies to make separate Fab and Fc fragments and subsequently to purify the Fab using Protein A agarose.

Features of the Fab Preparation Kit:

High capacity—use the standard size kit for fragmenting 0.25 to 4 mg IgG.
Enzyme-free digestion products—Immobilized Papain (beaded agarose resin) provides for control of the digestion reaction and complete removal of resulting antibody fragments from the proteolytic enzyme
Suitable for human and other species of IgG—the kit procedure is optimized for human, mouse and rabbit IgG. Papain-based digestion is effective for certain other species and subclasses of IgG (e.g., goat and pig), although the purification step requires that the antibody effectively binds to Protein A. (Note: for best results with mouse IgG1, use Part No. 44980.)
Provides ready-to-use Fab—digestion and final recovery of purified Fab fragments occurs in neutral pH sodium phosphate buffer, suitable for storage or immediate use in typical applications
Complete—kits include all reagents needed to prepare and purify antibody fragments
Fast—spin format greatly reduces sample processing time
Flexible—Protocols are included for multiple species and IgG subclasses, as well as sample size and concentration.
Efficient—enhanced yield and sample purity

These Fab Preparation Kits are suitable for human, rabbit, mouse and other species and subclasses of IgG. The papain antibody digestion reaction is performed in convenient disposable spin columns that allow efficient removal of the immobilized protease and maximum recovery of the IgG fragments. Also included in the kits are Thermo Scientific NAb Protein A Spin Columns and buffers to efficiently purify the resulting fragments. Protein A binds the Fc fragments and undigested IgG, allowing the pure Fab fragments to be recovered in the flow-through fraction. The kits also include Thermo Scientific Zeba Desalting Spin Columns for preparing the IgG sample quickly without dilution instead of utilizing time-consuming dialysis steps.

The kits use papain, a nonspecific thiol-endopeptidase, to enzymatically cleave whole IgG just above the hinge region to create two separate Fab fragments and one Fc fragment per antibody molecule. Because the papain protease is supplied in immobilized form as beaded agarose resin, the digestion reaction is easily stopped by removing the resin from the IgG solution; the result is digest products that are enzyme-free.

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Pierce™ Fab Micro Preparation Kit
Pierce™ F(ab')2 Preparation Kit
Pierce™ Mouse IgG1 Fab and F(ab')2 Preparation Kit

Trypsin, TPCK Treated Thermo Scientific™

Thermo Scientific Pierce TPCK Trypsin is a serine endoprotease that is applicable to amino acid analysis and protein sequencing, mapping and structural studies; the immobilized form allows sample separation after treatment.

Features of Thermo Scientific Pierce TPCK Trypsin

• Cleaves at carboxyl side of arginine and lysine residues
• Digestion conditions: pH 7.5-9.0, 37°

Trypsin has a wide range of applications including amino acid analysis and protein sequencing, mapping and structural studies. Enzymes such as trypsin and chymotrypsin have become important tools in sequencing studies since they are highly selective in their cleavage of peptide bonds. Trypsin cleaves only those peptide bonds in which the carboxyl group is contributed by a lysine or an arginine residue, regardless of the length or amino acid sequence of the chain. Our Immobilized TPCK Trypsin can be substituted for free trypsin in many application and is advantageous because it minimizes autolysis, eliminates contamination of a sample with the protease and allows control of the digestion by removing the trypsin. Immobilized trypsin is also more stable against heat-induced denaturation, resulting in longer maintenance of activity.

Applications:
• Removal of adherent cells from tissue culture flasks
• Preparing tryptic fragments for Edman degradation sequencing
• Immobilized trypsin can be used to purify soybean trypsin inhibitor
• Use our Pierce Trypsin, MS Grade (Part No. 90057) for mass spectrometry workflows

Trypsin is a 23.8kDa pancreatic serine endoprotease derived from trypsinogen, an inactive precursor zymogen, after enzymatic removal of an n-terminal leader sequence by enterokinase. Once some trypsin has been formed it can catalyze the conversion of more trypsinogen into its catalytically-active form. Trypsin is treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) to inhibit contaminating chymotrypsin activity without affecting trypsin activity.

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Immobilized Trypsin, TPCK Treated (Agarose Resin)

Immobilized Pepsin (Agarose Resin) Thermo Scientific™

Thermo Scientific Pierce Immobilized Pepsin Agarose consists of an acidic endopeptidase (pepsin) that has been immobilized onto beaded agarose resin to enable generation and purification of Fab and F(ab')2 fragments from antibodies.

Pepsin is a nonspecific acidic endopeptidase produced in an inactive precursor form (pepsinogen) in the mucosal lining of the stomach of vertebrates. There are several pepsins, each with a molecular weight of 35,000 and an optimum pH of 1-3; at pH >6, the enzyme is permanently inactivated. Pepsin has broad substrate specificity. It cleaves proteins preferentially at carboxylic groups of aromatic amino acids such as phenylalanine, tryptophan and tyrosine. It will not cleave at bonds containing valine, alanine or glycine. Cleavage at other residues occurs with varying efficiency.

Pepsin and other proteolytic enzymes are used in the laboratory analysis of various proteins. Pepsin and papain are often used to generate antibody fragments. Papain is used to cleave antibodies into Fab fragments, which contain a single antigen-binding domain. Pepsin is used to generate F(ab')2 fragments by removing only that portion of the Fc domain beyond the hinge region. The resulting F(ab')2 fragments are composed of two antibody-binding Fab' fragments connected by disulfides in the hinge region.

Immobilized pepsin can be substituted for free pepsin in any application, and is advantageous because it virtually eliminates autolysis, eliminates contamination of a sample with the protease and allows control of the digestion by removing the pepsin. Immobilized pepsin is also more stable against heat-induced denaturation, resulting in longer maintenance of activity. The F(ab')2 Preparation Kit contains immobilized pepsin, other necessary reagents and an optimized protocol for human, rabbit and mouse antibody digestion.

Pierce™ F(ab')2 Preparation Kit Thermo Scientific™

The Thermo Scientific Pierce F(ab')2 Preparation Kit uses immobilized pepsin protease to selectively digest whole human and other IgG antibodies to make F(ab')2 fragments that retain antigen binding activity.

Features of the F(ab')2 Micro Preparation Kit:

Enzyme-free digestion products—Immobilized Pepsin (beaded agarose resin) provides for control of the digestion reaction and complete removal of resulting antibody fragments from the proteolytic enzyme
Suitable for human and other species of IgG—the kit procedure is optimized for human, rabbit and mouse IgG. Pepsin-based digestion is effective for other species and subclasses of IgG, although the purification step requires that the antibody effectively binds to Protein A. (Note: for best results with mouse IgG1, use Part No. 44980.)
Complete—kits include all reagents needed to prepare and purify antibody fragments
Fast—spin format greatly reduces sample processing time
Flexible—protocols are included for multiple species and IgG subclasses, as well as sample size and concentration.
Efficient—enhanced yield and sample purity

These F(ab')2 Preparation Kits are suitable for human, rabbit, mouse and other species and subclasses of IgG except mouse IgG1. The antibody digestion reaction with pepsin agarose is performed in convenient disposable spin columns that allow efficient removal of the immobilized protease and maximum recovery of the IgG fragments. Also included in the kits are Thermo Scientific NAb Protein A Spin Columns and buffers to efficiently purify the resulting fragments. Protein A binds the Fc fragments and undigested IgG, allowing the pure F(ab')2 fragments to be recovered in the flow-through fraction. The kits also include Thermo Scientific Zeba Desalting Spin Columns for preparing the IgG sample quickly without dilution instead of utilizing time-consuming dialysis steps.

The kits use pepsin, a nonspecific endopeptidase, to enzymatically digest the Fc portion of whole IgG to yield the fragment known as F(ab')2. This fragment is composed of a pair of Fab' units connected by two disulfide bonds. Because the pepsin protease is supplied in immobilized form as beaded agarose resin, the digestion reaction is easily stopped by removing the resin from the IgG solution; the result is digest products that are enzyme-free.

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Pierce™ F(ab')2 Micro Preparation Kit
Pierce™ Fab Micro Preparation Kit
Pierce™ Mouse IgG1 Fab and F(ab')2 Micro Preparation Kit

Pierce™ F(ab')2 Micro Preparation Kit Thermo Scientific™

The Thermo Scientific Pierce F(ab')2 Micro Preparation Kit uses immobilized pepsin protease to selectively digest whole human and other IgG antibodies to make F(ab')2 fragments that retain antigen binding activity.

Features of the F(ab')2 Micro Preparation Kit:

Ideal when sample size is limited—Use the micro kit for 25 to 250 µg IgG.
Enzyme-free digestion products—Immobilized Pepsin (beaded agarose resin) provides for control of the digestion reaction and complete removal of resulting antibody fragments from the proteolytic enzyme
Suitable for human and other species of IgG—the kit procedure is optimized for human, rabbit and mouse IgG. Pepsin-based digestion is effective for other species and subclasses of IgG, although the purification step requires that the antibody effectively binds to Protein A. (Note: for best results with mouse IgG1, use Part No. 44980.)
Complete—kits include all reagents needed to prepare and purify antibody fragments
Fast—spin format greatly reduces sample processing time
Flexible—protocols are included for multiple species and IgG subclasses, as well as sample size and concentration.
Efficient—enhanced yield and sample purity

These F(ab')2 Preparation Kits are suitable for human, rabbit, mouse and other species and subclasses of IgG except mouse IgG1. The antibody digestion reaction with pepsin agarose is performed in convenient disposable spin columns that allow efficient removal of the immobilized protease and maximum recovery of the IgG fragments. Also included in the kits are Thermo Scientific NAb Protein A Spin Columns and buffers to efficiently purify the resulting fragments. Protein A binds the Fc fragments and undigested IgG, allowing the pure F(ab')2 fragments to be recovered in the flow-through fraction. The kits also include Thermo Scientific Zeba Desalting Spin Columns for preparing the IgG sample quickly without dilution instead of utilizing time-consuming dialysis steps.

The kits use pepsin, a nonspecific endopeptidase, to enzymatically digest the Fc portion of whole IgG to yield the fragment known as F(ab')2. This fragment is composed of a pair of Fab' units connected by two disulfide bonds. Because the pepsin protease is supplied in immobilized form as beaded agarose resin, the digestion reaction is easily stopped by removing the resin from the IgG solution; the result is digest products that are enzyme-free.

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Pierce™ F(ab')2 Preparation Kit
Pierce™ Fab Micro Preparation Kit
Pierce™ Mouse IgG1 Fab and F(ab')2 Micro Preparation Kit

Pierce™ Fab Micro Preparation Kit Thermo Scientific™

Thermo Scientific Pierce Fab Micro Preparation Kit uses immobilized papain protease to digest human or mouse IgG antibodies to make separate Fab and Fc fragments and subsequently to purify the Fab using Protein A agarose.

Features of the Fab Micro Preparation Kit:

High capacity—use the micro kit for 25 to 250 µg IgG.
Enzyme-free digestion products—Immobilized Papain (beaded agarose resin) provides for control of the digestion reaction and complete removal of resulting antibody fragments from the proteolytic enzyme
Suitable for human and other species of IgG—the kit procedure is optimized for human, mouse and rabbit IgG. Papain-based digestion is effective for certain other species and subclasses of IgG (e.g., goat and pig), although the purification step requires that the antibody effectively binds to Protein A. (Note: for best results with mouse IgG1, use Part No. 44980.)
Provides ready-to-use Fab—digestion and final recovery of purified Fab fragments occurs in neutral pH sodium phosphate buffer, suitable for storage or immediate use in typical applications
Complete—kits include all reagents needed to prepare and purify antibody fragments
Fast—spin format greatly reduces sample processing time
Flexible—Protocols are included for multiple species and IgG subclasses, as well as sample size and concentration.
Efficient—enhanced yield and sample purity

These Fab Preparation Kits are suitable for human, rabbit, mouse and other species and subclasses of IgG. The papain antibody digestion reaction is performed in convenient disposable spin columns that allow efficient removal of the immobilized protease and maximum recovery of the IgG fragments. Also included in the kits are Thermo Scientific NAb Protein A Spin Columns and buffers to efficiently purify the resulting fragments. Protein A binds the Fc fragments and undigested IgG, allowing the pure Fab fragments to be recovered in the flow-through fraction. The kits also include Thermo Scientific Zeba Desalting Spin Columns for preparing the IgG sample quickly without dilution instead of utilizing time-consuming dialysis steps.

The kits use papain, a nonspecific thiol-endopeptidase, to enzymatically cleave whole IgG just above the hinge region to create two separate Fab fragments and one Fc fragment per antibody molecule. Because the papain protease is supplied in immobilized form as beaded agarose resin, the digestion reaction is easily stopped by removing the resin from the IgG solution; the result is digest products that are enzyme-free.

Related Products
Pierce™ Fab Preparation Kit
Pierce™ F(ab')2 Micro Preparation Kit
Pierce™ Mouse IgG1 Fab and F(ab')2 Micro Preparation Kit

Immobilized Ficin (Agarose Resin) Thermo Scientific™

Thermo Scientific Pierce Immobilized Ficin consists of a sulfhydryl-specific protease (ficin) that has been immobilized onto beaded agarose resin to enable controlled antibody fragmentation, especially of mouse IgG1.

Features of Pierce Immobilized Ficin:

• Can generate both Fab and F(ab')2 fragments from mouse IgG1
• Reaction can be easily controlled
• Antibody fragments are free of autodigestion products
• Ficin contamination into antibody fragments is eliminated

Ficin, which is similar to papain, is a nonspecific, sulfhydryl protease isolated from fig latex. The molecular weight of Ficin is approximately 25,000 and it is effective at pH 4-9.5 with an optimum pH of 6.5. When IgG molecules are incubated with ficin in the presence of cysteine, one or more peptide bonds in the hinge region are split, producing either Fab fragments or F(ab')2 fragments. Mouse IgG1 is notably difficult to digest into functional Fab or F(ab')2 fragments using papain or pepsin, however, yields and immunoreactivity are better when digesting mouse IgG1 with ficin. Immobilized Ficin was specifically designed for cleavage of mouse IgG1 into or Fab or F(ab')2 fragments, and is advantageous because it virtually eliminates autolysis, eliminates contamination of a sample with the protease and allows control of the digestion by removing the ficin or controlling the flow of sample over an immobilized ficin column.

Immobilized ficin is more stable against heat-induced denaturation than the free enzyme, resulting in longer maintenance of activity. The Fab and F(ab')2 Preparation Kit contains immobilized ficin, other necessary reagents and an optimized protocol for mouse IgG1 digestion. Ficin will generate mouse IgG1 F(ab')2 and Fab fragments in the presence of 1 mM cysteine and 10 mM cysteine, respectively. Fragment generation from other species and isotypes is also possible through modification of the cysteine concentration and other digestion parameters.

Immobilized Papain (Agarose Resin) Thermo Scientific™

Thermo Scientific Pierce Immobilized Papain consists of a cysteine-endopeptidase (papain) that has been immobilized onto beaded agarose resin to enable generation and purification of Fab fragments from antibodies.

Papain is a nonspecific, cysteine-endopeptidase isolated from papaya latex that is used in a wide variety of applications. When IgG molecules are incubated with papain in the presence of cysteine, one or more peptide bonds in the hinge region are split, producing three fragments of similar size: two Fab fragments and one Fc fragment. The Fc fragments can be separated from the Fab fragments using immobilized Protein A or by ion exchange chromatography.

The 50 kilodalton Fc fragment is often used for determining specificity of the Fc receptors without interference from antigen binding. Fab fragments are useful in immunohistochemical studies because the fragments penetrate the tissue better than intact IgG. Also, nonspecific binding from Fc receptors is avoided by using only the Fab portion of the immunoglobulin.

Papain can also can be used to generate F(ab')2 fragments. To prepare F(ab')2 fragments, the papain is first activated with 10 mM cysteine, which is then removed via gel filtration. If no cysteine is present during the papain digestion, F(ab')2 fragments can be generated. These fragments are often inconsistent and reproducibility is a problem.

Immobilized papain can be substituted for free papain in almost any application, and is advantageous because it virtually eliminates autolysis, eliminates contamination of a sample with the protease and allows control of the digestion by removing the papain or controlling the flow of sample over an immobilized papain column. Immobilized papain is also more stable against heat-induced denaturation, resulting in longer maintenance of activity. The ImmunoPure Fab Preparation Kit contains immobilized papain, other necessary reagents and an optimized protocol for human, rabbit and mouse antibody digestion.

Pierce™ Mouse IgG1 Fab and F(ab')2 Micro Preparation Kit Thermo Scientific™

The Thermo Scientific Pierce Mouse IgG1 Fab and F(ab')2 Micro Preparation Kit uses immobilized ficin protease to efficiently digest mouse IgG1 into Fab or F(ab')2 fragments, depending on the chosen reaction conditions.

Features of the Mouse IgG1 Fab and F(ab')2 Micro Preparation Kit:

Capacity—kit provides for fragmenting 25 to 250 µg IgG.
Complete—kit includes all reagents needed to prepare and purify antibody fragments
Enzyme-free digestion products—Immobilized Ficin (beaded agarose resin) provides for control of the digestion reaction and complete removal of resulting antibody fragments from the proteolytic enzyme
Optimized for mouse IgG1—the kit procedure is optimized for mouse IgG1; the only effective method for this IgG species and subclass (see

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Pierce™ Mouse IgG1 Fab and F(ab')2 Preparation Kit
Pierce™ F(ab')2 Micro Preparation Kit
Pierce™ Fab Micro Preparation Kit

Immobilized Trypsin, TPCK Treated (Agarose Resin) Thermo Scientific™

Thermo Scientific Pierce Immobilized TPCK Trypsin is a serine endoprotease that is applicable to amino acid analysis and protein sequencing, mapping and structural studies; the immobilized form allows sample separation after treatment.

Features of Thermo Scientific Pierce Immobilized TPCK Trypsin

• Cleaves at carboxyl side of arginine and lysine residues
• Digestion conditions: pH 7.5-9.0, 37°
• Immobilized for ease of separation from cleavage products or affinity purification of Trypsin Inhibitors

Trypsin has a wide range of applications including amino acid analysis and protein sequencing, mapping and structural studies. Enzymes such as trypsin and chymotrypsin have become important tools in sequencing studies since they are highly selective in their cleavage of peptide bonds. Trypsin cleaves only those peptide bonds in which the carboxyl group is contributed by a lysine or an arginine residue, regardless of the length or amino acid sequence of the chain. Our Immobilized TPCK Trypsin can be substituted for free trypsin in many application and is advantageous because it minimizes autolysis, eliminates contamination of a sample with the protease and allows control of the digestion by removing the trypsin. Immobilized trypsin is also more stable against heat-induced denaturation, resulting in longer maintenance of activity.

Applications:
• Removal of adherent cells from tissue culture flasks
• Preparing tryptic fragments for Edman degradation sequencing
• Immobilized trypsin can be used to purify soybean trypsin inhibitor
• Use our Pierce Trypsin, MS Grade (Part No. 90057) for mass spectrometry workflows

Trypsin is a 23.8kDa pancreatic serine endoprotease derived from trypsinogen, an inactive precursor zymogen, after enzymatic removal of an n-terminal leader sequence by enterokinase. Once some trypsin has been formed it can catalyze the conversion of more trypsinogen into its catalytically-active form. Trypsin is treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) to inhibit contaminating chymotrypsin activity without affecting trypsin activity.

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Staphylococcus aureus V-8 Protease Thermo Scientific™

Thermo Scientific Staphylococcus aureus V-8 Protease also known as endoproteinase Glu-C, has two pH optima, one at pH 4.0 and the other at pH 7.8, and is specific for cleavage at the carboxy terminus of glutamic acid and aspartic acid residues. The protease is specific only for glutamic digestion in buffers which do not contain phosphate at either pH optimum. Phosphate buffers at pH 7.8 will facilitate digestion at both glutamic and aspartic acid residues.

Features of Staphylococcus aureus V-8 Protease:

Specificity—Specificity for glutamic acid is achieved in ammonium bicarbonate, pH 7.8 or ammonium acetate, pH 4
Activity—Enzyme is active in the presence of many denaturing agents such as SDS, urea and guanidine-HCl

S. aureus V-8 protease protease is very stable, being able to retain its activity even in 6 M urea, 5.5 M guanidine·HCl, 0.5% SDS, or 0.5% SDS + 6 M urea at 0°C, pH 7.6. This is related to the fact that the native structure of the protease is stabilized mainly by electrostatic interactions, and not by hydrogen bonding and β-structures.

There are two major problems with any proteolytic digestion. The first problem is that the reaction must be quenched to stop the digestion, thus diluting out the sample. The second problem is that the sample is contaminated with the protease and possibly autodigested protease fragments. These problems are surmounted with immobilized V-8 protease. With immobilized proteases, no quenching is necessary and there are no problems with protease contamination of the sample or autodigestion of the protease. Simply separate the sample solution from the agarose matrix and these problems are eliminated.

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