Amine-Reactive Probe Labeling Experimental Protocol
Product Manager：Harrison Michael
Amine-reactive probe labeling techniques are widely used for the labeling and tracking of biomolecules, especially in the modification of proteins and nucleic acids. By covalently attaching fluorescent dyes, biotin, or other probes to the amine groups of target molecules, researchers can trace and analyze molecule behavior, localization, and interactions with other molecules. This protocol provides a step-by-step guide for labeling proteins (such as IgG antibodies) or amine-modified oligonucleotides with amine-reactive probes. This method is valuable for protein analysis, nucleic acid probe preparation, and biomolecular tracking applications.
Types of Amine-Reactive Labeling Reagents
Common amine-reactive reagents include:
• Active Esters: Such as Succinimidyl Ester (SE), Sulfo-Succinimidyl Ester (SSE), Tetrafluorophenyl Ester (TFP), and Sulfo-Dichlorophenyl Ester (SDP), which form stable amide bonds with amine groups in proteins or nucleic acids.
• Isothiocyanates (ITC): Frequently used for protein labeling; however, the thiourea bond formed is relatively weak, which may affect the stability of the labeled molecule.
• Sulfonyl Chlorides (SC): These react rapidly but must be handled under low-temperature conditions to maintain stability.
Among these, SDP ester is more resistant to hydrolysis in aqueous solutions compared to other active esters, helping to improve labeling efficiency and control the reaction process. Other reagents suitable for amine reactions include dichlorotriazines, aryl halides, and acyl azides. This protocol uses amine-reactive probes dissolved in inorganic solvents, suitable for both aqueous and non-aqueous labeling experiments.
1. Protein Labeling Protocol
Required Materials
• Protein Sample: Suitable for labeling most proteins or peptides containing amine groups, using IgG antibody as an example. A sample concentration above 2 mg/mL is recommended for better reaction efficiency.
• Labeling Dye: TFP ester, SDP ester, or succinimidyl ester dyes are recommended for forming stable amide bonds during labeling.
• Solvent: Most dyes need to be dissolved in high-purity anhydrous DMF or DMSO to ensure solubility and prevent incomplete reactions caused by moisture. Note: Do not use DMSO for sulfonyl chloride dyes.
• Buffer: Use sodium bicarbonate buffer at pH 8.3 or 9.0 to ensure the amine groups on the protein remain unprotonated. Avoid buffers with primary amine groups such as Tris.
• Quenching Agent (Optional): 1.5 M hydroxylamine solution (pH 8.5) to remove unbound dye and terminate the reaction. Prepare fresh before use.
Experimental Steps
1. Dissolve 10 mg of protein in 1 mL of 0.1 M sodium bicarbonate buffer, maintaining a concentration between 5-20 mg/mL.
2. Dissolve the dye in anhydrous DMF or DMSO at a concentration of 10 mg/mL.
3. While stirring the protein solution, slowly add the dye solution, aiming for a total dye amount of about 0.5-1 mg.
4. Allow the reaction to proceed at room temperature for 1 hour. For sulfonyl chloride reagents, perform the reaction at 4°C for stability.
5. If quenching the reaction and removing unbound dye, add 0.1 mL of 1.5 M hydroxylamine solution and continue stirring for 1 hour.
6. Purify the labeled protein using a gel filtration column, such as Sephadex™ G-25, to remove unreacted dye.
2. Amine-Modified Oligonucleotide Labeling Protocol
Required Materials
• Oligonucleotide Sample: Typically a 5'-amine-modified oligonucleotide. This protocol uses a 100 μg sample.
• Labeling Dye: Succinimidyl ester dyes like BODIPY® or Alexa Fluor® for high brightness and stability.
• Solvent: Dissolve the dye in anhydrous DMSO.
• Buffer: Use a borate buffer at pH 8.5 to optimize labeling conditions for oligonucleotides.
Experimental Steps
1. Dissolve the amine-modified oligonucleotide in borate buffer to prepare for labeling.
2. Dissolve the dye in anhydrous DMSO and quickly add it to the oligonucleotide solution.
3. Allow the reaction to proceed at room temperature for 1 hour, ensuring thorough mixing.
4. Purify the labeled oligonucleotide by high-performance liquid chromatography (HPLC) or affinity chromatography to remove unbound dye.
This protocol provides stable and efficient amine-reactive labeling steps, applicable to the modification and analysis of proteins and oligonucleotides. Aladdin scientific offers high-quality reagents for amine-reactive probe labeling protocols. Feel free to visit our website for inquiries and purchases.
Aladdin：https://www.aladdinsci.com/