Maleimide Rapid Labeling Kit (100 µg Scale)

The Maleimide Rapid Labeling Kit uses a simple and quick process to label proteins with maleimide (SMCC) groups, conferring sulfhydryl reactivity that enables conjugation with sulfhydryl group-containing molecules such as antibodies. The conjugate can be used immediately in applications such as Western blot, ELISA, and Immunohistochemistry (IHC). Sulfo-SMCC is a crosslinker that contains sulfonated N‑hydroxysuccinimide (NHS) ester and maleimide reactive groups. NHS esters target primary amines at pH 7.0–8.5 to form amide bonds, as shown in reaction (1) of Figure 1, while maleimides react with sulfhydryl groups at pH 6.5–7.5 to form stable thioether bonds, as shown in reaction (2) of Figure 1. The cyclohexane ring in the spacer arm of Sulfo‑SMCC decreases the rate of hydrolysis of the maleimide group compared to similar reagents that do not contain this ring. This feature enables proteins that have been maleimide‑labeled with Sulfo‑SMCC to be lyophilized and stored for later use in conjugation reactions. 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 Maleimide Rapid Labeling Kit (100 µg Scale) (S1510889) Labeling Principle SchematicRequired materials not supplied1. Microcentrifuge capable of 1,000 xg.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. Sulfo-SMCC 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. 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.4. Do not reuse the purification resin.5. During operation, always wear a lab coat, disposable gloves, and protective equipment.6. 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 Sulfo-SMCC (10 mg/mL): Add 200 μL of DMSO to the Sulfo-SMCC. Vortex or pipet up and down until the solution is homogeneous. Note: If the dissolved Sulfo-SMCC is not used immediately, aliquot the remaining solution and store at –20°C for up to two months.1.3 Dissolve or dilute the protein to be labeled with 1×Conjugation Buffer to adjust the concentration to 1–10 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 Sulfo-SMCC may be appropriately increased.Table 1. Recommended Sulfo-SMCC : Proteins at Different Concentrations Molar Ratios (MR)Sample typeSample concentrationRecommended Sulfo-SMCC molar excessIgG antibody1mg/mL25X-40XIgG antibody2-5mg/mL15X-25XPurified protein1–4 mg/mL20XPurified protein5–10 mg/mL15X2.2 Use equation 1 to calculate the appropriate volume of Sulfo-SMCC solution to use:Equation 1(μg protein×MR×Sulfo SMCC MW)/(protein MW×10)= μL Sulfo-SMCC to add to samplewhere µg protein is the mass of protein you want to label, MR is the Sulfo-SMCC : protein molar ratio from Table 1, Sulfo-SMCC MW is the molecular weight of the Sulfo-SMCC in Da (MW 436.37 Da), protein MW is the molecular weight of your protein in Da, and 10 is the concentration of the Sulfo-SMCC solution. For example, to label 100 µg of IgG (MW 150,000 Da):(100×25×436.37)/（150000×10）=0.73 μL3. SMCC Labeling Reaction3.1 Prepare the protein to be labeled. Calculate the required amount of Sulfo-SMCC 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.Note: During the incubation period, proceed to steps 3 below, to prepare a spin column for the purification of the labeled protein.4. Prepare the spin column4.1 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.4.2 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.4.3 Repeat step 4.2 for two additional times. Add 1×Conjugation Buffer for the third wash but wait to centrifuge until immediately prior to proceeding to step.4.4 After the third wash check there is no solution in the column, if required briefly centrifuge until no solution remains.5. Purification of SMCC-Labeled Protein5.1 Insert prepared desalting column into the new Collection Tube and apply the entire sample from step 3.1 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.5.2 Centrifuge the desalting column at 1000×g for 2 minutes. The flow-through in the collection tube is the desired SMCC-labeled protein.6. Strorage6.1 Proceed immediately to the next step for crosslinking with the sulfhydryl-containing protein (not provided with this kit). Alternatively, lyophilize the SMCC-labeled protein as soon as possible for future use.

Storage Temperature: Store at 2-8°C;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:

S1510889-10reactions

Country of Origin:

China

Product Size:

10reactions

Harmonized Tariff Code:

2931.90.90