Sulfhydryl Rapid Labeling Kit (100 µg Scale)

IntroductionSulfhydryl Rapid Labeling Kit uses a simple and quick process to introduce free sulfhydryl groups (–SH) into any molecule containing primary amines (NH₂). Proteins, antibodies, and other molecules labeled with this kit can rapidly, efficiently, and spontaneously conjugate with maleimide-containing biomolecules under mild conditions. The conjugates can be used in applications such as Western blot, ELISA, and Immunohistochemistry (IHC).This kit uses SATA for the introduction of sulfhydryl groups. SATA is a heterobifunctional crosslinker that contains a sulfonated N-hydroxysuccinimide (NHS) ester and a protected sulfhydryl group. The NHS ester reacts with primary amines at pH 7–8.5 to form stable covalent amide bonds, as shown in reaction (1) of Figure 1. The protected sulfhydryl group can be exposed when needed for the conjugation reaction, as shown in reaction (2) of Figure 1, and then reacts with maleimide groups to form stable thioether bonds.The advantage of SATA is that the sulfhydryl group is introduced in a protected form. The modified protein can be stored for extended periods without deprotection. When the next crosslinking reaction is required, hydroxylamine is used to expose the sulfhydryl group, allowing the conjugation reaction progress to be controlled. This kit provides all necessary reagents and purification columns sufficient for 10 labeling reactions, each using 100 μg of antibody, as well as a detailed step-by-step protocol. Fig. 1 Sulfhydryl Rapid Labeling Kit (100 µg Scale) (S1510886) Labeling Principle SchematicRequired materials not supplied1. Microcentrifuge capable of 1,000 × g.2. Desired antibody for labeling (free of BSA or any carrier protein).3. PBS buffer (pH 7.2-7.4).Important1. Any primary amines in the IgG solution (e.g., Tris or glycine) will interfere with conjugation. It is recommended to dissolve the molecule to be labeled in 1×Conjugation Buffer.2. SATA is easily inactivated by moisture-induced hydrolysis. Care must be taken to seal the container tightly and keep it dry during storage. After preparing the stock solution in DMSO, it can be aliquoted and stored at -20°C for up to two months. Storage at -80°C allows for even longer preservation.3. Hydroxylamine is hygroscopic. Keep it dry during storage. Allow it to equilibrate to room temperature (approximately 25°C) before opening the bottle for weighing.4. Before formal experimentation, the reagents must be returned to room temperature and centrifuged before use. Both lower temperatures and reagent residue on the centrifuge tube walls may impair conjugation efficiency.5. Do not reuse the purification resin.6. During operation, always wear a lab coat, disposable gloves, and protective equipment.7. All products are for research use only.Instruction for use1. Preparations1.1 Preparation of 1 × Conjugation Buffer: Dilute the 10 × Conjugation Buffer solution 10-fold with PBS (pH 7.2-7.4) to prepare 1 × Conjugation Buffer.1.2 Preparation of SATA (4 mg/mL): Add 500 μL of DMSO to the SATA. Vortex or pipet up and down until the solution is homogeneous. Note: If the dissolved SATA is not used immediately, aliquot the remaining solution and store at -20°C for up to two months. Storage at -80°C allows for even longer preservation.1.3 Dissolve or dilute the protein to be labeled with 1 × Conjugation Buffer to adjust the concentration to 0.5–5 mg/mL. If the protein to be labeled is not in a salt-free state or is already in a solution, it is necessary to exchange the buffer into 1 × Conjugation Buffer by concentration or dialysis.2. Determine the Reaction Ratio2.1 The labeling reaction should be performed according to the recommended ratios in Table 1, based on the protein concentration. If the labeling efficiency is not satisfactory, the ratio of SATA may be appropriately increased.Table 1. Recommended SATA : Proteins at Different Concentrations Molar Ratios (MR)Sample typeSample concentrationRecommended SATA molar excessProtein0.5mg/mL30× - 40×Protein0.5-5mg/mL15× - 30×2.2 Use equation 1 to calculate the appropriate volume of SATA solution to use:Equation 1(μg protein×MR×SATA MW)/(protein MW×4)= μL SATA to add to samplewhere µg protein is the mass of protein you want to label, MR is the SATA : protein molar ratio from Table 1, SATA MW is the molecular weight of the SATA in Da (MW 231.22 Da), protein MW is the molecular weight of your protein in Da, and 4 is the concentration of the SATA solution. For example, to label 100 µg of IgG (MW 150,000 Da), MR is 30×:(100×30×231.22)/（150000×4）=1.16 μL3. SATA Labeling Reaction3.1 Prepare the protein to be labeled. Calculate the required amount of SATA according to Table 1 and Equation 1. Mix the two components and gently vortex to mix thoroughly.3.2 Incubate at room temperature (approximately 25°C) for 30 minutes or at 4°C for 2 hours. Due to the small sample volume, a micro-vortex mixer is recommended at 1500 rpm.4. De-protect the latent sulfhydryl4.1 Preparation of Hydroxylamine HCl (40 mg/mL): Allow the Hydroxylamine HCl to equilibrate to room temperature before opening the bottle. Weigh an appropriate amount of Hydroxylamine HCl and dissolve it in 1 × Conjugation Buffer to prepare a 40 mg/mL Hydroxylamine HCl solution. Adjust the pH to 7.2 using NaOH. Note: Hydroxylamine HCl must be prepared fresh immediately before use.4.2 Add Hydroxylamine HCl at 1/10 of the reaction volume from step 3.1. For example, for a reaction volume of 0.5 mL, add 50 µL of hydroxylamine hydrochloride and mix gently by vortexing.4.3 Incubate at room temperature (approximately 25°C) for 2 hours. Due to the small sample volume, a micro-vortex mixer is recommended at 1500 rpm.Note: During the incubation period, proceed to steps 5 below, to prepare a spin column for the purification of the labeled protein.5. Prepare the spin column5.1 Loosen the cap on a spin column, twist the tab off of the bottom, then place the column into a collection tube. Centrifuge the desalting column assembly at 1,000 x g for 2 minutes to remove the storage solution. Discard the flowthrough, then place the column back into the collection tube.Note: When using a fixed-angle rotor, place a mark on the side of the column that faces away from the rotor center. For all subsequent centrifugation steps, place the column in the microcentrifuge with the mark facing away from the rotor center.5.2 Add 500 μL of 1 × Conjugation Buffer, then centrifuge the desalting column assembly at 1,000 x g for 2 minutes to equilibrate the column.5.3 Repeat step 5.2 for two additional times. Add 1 × Conjugation Buffer for the third wash but wait to centrifuge until immediately prior to proceeding to step.5.4 After the third wash check there is no solution in the column, if required briefly centrifuge until no solution remains.6. Purification of sulfhydryl-Labeled Protein6.1 Insert prepared desalting column into the new Collection Tube and apply the entire sample from step 4.2 to the column. Cap the desalting column, taking care to screw the cap on gently. The loading volume range for the desalting column is 70 µL to 120 µL. If the volume is insufficient, top up with 1 × Conjugation Buffer.6.2 Centrifuge the desalting column at 1000 × g for 2 minutes. The flow-through in the collection tube is the desired sulfhydryl-labeled protein.7. Strorage7.1 Proceed immediately to the next step of conjugation with maleimide-containing protein (not provided with this kit), or perform deprotection of the sulfhydryl groups when the next reaction is required.

Specifications & Purity: IntroductionSulfhydryl Rapid Labeling Kit uses a simple and quick process to introduce free sulfhydryl groups (–SH) into any molecule containing primary amines (NH₂). Proteins, antibodies, and other molecules labeled with this kit can rapidly, efficiently, and spontaneously conjugate with maleimide-containing biomolecules under mild conditions. The conjugates can be used in applications such as Western blot, ELISA, and Immunohistochemistry (IHC).This kit uses SATA for the introduction of sulfhydryl groups. SATA is a heterobifunctional crosslinker that contains a sulfonated N-hydroxysuccinimide (NHS) ester and a protected sulfhydryl group. The NHS ester reacts with primary amines at pH 7–8.5 to form stable covalent amide bonds, as shown in reaction (1) of Figure 1. The protected sulfhydryl group can be exposed when needed for the conjugation reaction, as shown in reaction (2) of Figure 1, and then reacts with maleimide groups to form stable thioether bonds.The advantage of SATA is that the sulfhydryl group is introduced in a protected form. The modified protein can be stored for extended periods without deprotection. When the next crosslinking reaction is required, hydroxylamine is used to expose the sulfhydryl group, allowing the conjugation reaction progress to be controlled. This kit provides all necessary reagents and purification columns sufficient for 10 labeling reactions, each using 100 μg of antibody, as well as a detailed step-by-step protocol. Fig. 1 Sulfhydryl Rapid Labeling Kit (100 µg Scale) (S1510886) Labeling Principle SchematicRequired materials not supplied1. Microcentrifuge capable of 1,000 × g.2. Desired antibody for labeling (free of BSA or any carrier protein).3. PBS buffer (pH 7.2-7.4).Important1. Any primary amines in the IgG solution (e.g., Tris or glycine) will interfere with conjugation. It is recommended to dissolve the molecule to be labeled in 1×Conjugation Buffer.2. SATA is easily inactivated by moisture-induced hydrolysis. Care must be taken to seal the container tightly and keep it dry during storage. After preparing the stock solution in DMSO, it can be aliquoted and stored at -20°C for up to two months. Storage at -80°C allows for even longer preservation.3. Hydroxylamine is hygroscopic. Keep it dry during storage. Allow it to equilibrate to room temperature (approximately 25°C) before opening the bottle for weighing.4. Before formal experimentation, the reagents must be returned to room temperature and centrifuged before use. Both lower temperatures and reagent residue on the centrifuge tube walls may impair conjugation efficiency.5. Do not reuse the purification resin.6. During operation, always wear a lab coat, disposable gloves, and protective equipment.7. All products are for research use only.Instruction for use1. Preparations1.1 Preparation of 1 × Conjugation Buffer: Dilute the 10 × Conjugation Buffer solution 10-fold with PBS (pH 7.2-7.4) to prepare 1 × Conjugation Buffer.1.2 Preparation of SATA (4 mg/mL): Add 500 μL of DMSO to the SATA. Vortex or pipet up and down until the solution is homogeneous. Note: If the dissolved SATA is not used immediately, aliquot the remaining solution and store at -20°C for up to two months. Storage at -80°C allows for even longer preservation.1.3 Dissolve or dilute the protein to be labeled with 1 × Conjugation Buffer to adjust the concentration to 0.5–5 mg/mL. If the protein to be labeled is not in a salt-free state or is already in a solution, it is necessary to exchange the buffer into 1 × Conjugation Buffer by concentration or dialysis.2. Determine the Reaction Ratio2.1 The labeling reaction should be performed according to the recommended ratios in Table 1, based on the protein concentration. If the labeling efficiency is not satisfactory, the ratio of SATA may be appropriately increased.Table 1. Recommended SATA : Proteins at Different Concentrations Molar Ratios (MR)Sample typeSample concentrationRecommended SATA molar excessProtein0.5mg/mL30× - 40×Protein0.5-5mg/mL15× - 30×2.2 Use equation 1 to calculate the appropriate volume of SATA solution to use:Equation 1(μg protein×MR×SATA MW)/(protein MW×4)= μL SATA to add to samplewhere µg protein is the mass of protein you want to label, MR is the SATA : protein molar ratio from Table 1, SATA MW is the molecular weight of the SATA in Da (MW 231.22 Da), protein MW is the molecular weight of your protein in Da, and 4 is the concentration of the SATA solution. For example, to label 100 µg of IgG (MW 150,000 Da), MR is 30×:(100×30×231.22)/（150000×4）=1.16 μL3. SATA Labeling Reaction3.1 Prepare the protein to be labeled. Calculate the required amount of SATA according to Table 1 and Equation 1. Mix the two components and gently vortex to mix thoroughly.3.2 Incubate at room temperature (approximately 25°C) for 30 minutes or at 4°C for 2 hours. Due to the small sample volume, a micro-vortex mixer is recommended at 1500 rpm.4. De-protect the latent sulfhydryl4.1 Preparation of Hydroxylamine HCl (40 mg/mL): Allow the Hydroxylamine HCl to equilibrate to room temperature before opening the bottle. Weigh an appropriate amount of Hydroxylamine HCl and dissolve it in 1 × Conjugation Buffer to prepare a 40 mg/mL Hydroxylamine HCl solution. Adjust the pH to 7.2 using NaOH. Note: Hydroxylamine HCl must be prepared fresh immediately before use.4.2 Add Hydroxylamine HCl at 1/10 of the reaction volume from step 3.1. For example, for a reaction volume of 0.5 mL, add 50 µL of hydroxylamine hydrochloride and mix gently by vortexing.4.3 Incubate at room temperature (approximately 25°C) for 2 hours. Due to the small sample volume, a micro-vortex mixer is recommended at 1500 rpm.Note: During the incubation period, proceed to steps 5 below, to prepare a spin column for the purification of the labeled protein.5. Prepare the spin column5.1 Loosen the cap on a spin column, twist the tab off of the bottom, then place the column into a collection tube. Centrifuge the desalting column assembly at 1,000 x g for 2 minutes to remove the storage solution. Discard the flowthrough, then place the column back into the collection tube.Note: When using a fixed-angle rotor, place a mark on the side of the column that faces away from the rotor center. For all subsequent centrifugation steps, place the column in the microcentrifuge with the mark facing away from the rotor center.5.2 Add 500 μL of 1 × Conjugation Buffer, then centrifuge the desalting column assembly at 1,000 x g for 2 minutes to equilibrate the column.5.3 Repeat step 5.2 for two additional times. Add 1 × Conjugation Buffer for the third wash but wait to centrifuge until immediately prior to proceeding to step.5.4 After the third wash check there is no solution in the column, if required briefly centrifuge until no solution remains.6. Purification of sulfhydryl-Labeled Protein6.1 Insert prepared desalting column into the new Collection Tube and apply the entire sample from step 4.2 to the column. Cap the desalting column, taking care to screw the cap on gently. The loading volume range for the desalting column is 70 µL to 120 µL. If the volume is insufficient, top up with 1 × Conjugation Buffer.6.2 Centrifuge the desalting column at 1000 × g for 2 minutes. The flow-through in the collection tube is the desired sulfhydryl-labeled protein.7. Strorage7.1 Proceed immediately to the next step of conjugation with maleimide-containing protein (not provided with this kit), or perform deprotection of the sulfhydryl groups when the next reaction is required.

Grade: BioReagent

Storage Temperature: Store at 2-8°C;Protected from light;Room temperature;Store at -20°C;Do not freeze

Shipped in: Wet ice;Do not freeze

UPC:

41106000

Weight:

1.09 Ounces

HazmatClass:

No

Quantity:

1

Unit of Measurement:

ST

WeightUOM:

LB

MPN:

S1510886-10reactions

Country of Origin:

China

Product Size:

10reactions

Harmonized Tariff Code:

2931.90.90