What is the protocol for Atto 488 labeling?
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Protocol for Atto 488 Labeling
Atto 488 is a high-performance fluorescent label used for labeling biomolecules, particularly proteins and nucleic acids, enabling their detection and analysis in various biological assays. The following protocol outlines the general steps for Atto 488 labeling.
Materials Needed
- Atto 488 NHS Ester
- Buffer: 0.1 M Sodium Bicarbonate (pH 8.3)
- Protein or Nucleic Acid to be labeled
- Dialysis tubing or desalting column
- UV-Vis Spectrophotometer
Protocol Steps
- Dissolve Atto 488 NHS ester in anhydrous DMSO or DMF to make a 10 mM stock solution.
- Adjust the pH of the buffer (0.1 M Sodium Bicarbonate, pH 8.3) to ensure optimal labeling conditions.
- Mix the Atto 488 solution with the biomolecule solution. The typical molar ratio of dye to biomolecule is 1:1 to 10:1, depending on the desired degree of labeling.
- Incubate the reaction mixture for 1 hour at room temperature in the dark to prevent photobleaching of the dye.
- Remove unreacted dye by dialysis or using a desalting column against an appropriate buffer.
- Measure the degree of labeling (DOL) by UV-Vis spectrophotometry. Calculate the DOL using the absorbance of Atto 488 and the protein or nucleic acid.
Important Notes
- Always protect the Atto 488 dye from light to prevent photobleaching.
- The efficiency of labeling and the stability of the conjugate may vary depending on the biomolecule.
- Optimize the molar ratio of dye to biomolecule and reaction conditions based on specific applications.
- Ensure that the biomolecule to be labeled is in a buffer that does not contain primary amines (e.g., Tris, glycine) as they can react with the NHS ester.
Atto 488 labeling is a versatile technique for fluorescently tagging biomolecules for various applications in molecular biology and biochemistry. Following the outlined protocol and optimizing conditions based on specific needs will ensure successful labeling and accurate detection in assays.