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| 1mg | $338.00 | In Stock | 1 |
Alexa fluor 647 NHS ester can be used to label Alexa fluor 647 to the primary amines (R-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules[1].
References:
[1]. Martin Lehmann, et al. Multicolor Caged dSTORM Resolves the Ultrastructure of Synaptic Vesicles in the Brain. Angew Chem Int Ed Engl. 2015 Nov 2;54(45):13230-5.
Protocol
I. Protein Preparation
1) For optimal labeling, prepare a protein (antibody) concentration of 2 mg/mL.
2) The pH of the protein solution should be 8.5 ± 0.5. If the pH is below 8.0, adjust it using 1 M sodium bicarbonate.
3) Labeling efficiency will be significantly reduced if the protein concentration is below 2 mg/mL. For optimal labeling efficiency, a final protein concentration range of 2-10 mg/mL is recommended.
4) The protein must be in a buffer free of primary amines (such as Tris or glycine) and ammonium ions; otherwise, labeling efficiency will be affected.
2. Dye Preparation (using Alexa fluor 647 NHS ester as an example)
Add anhydrous DMSO to an Alexa fluor 647 NHS ester vial to prepare a 10 mM stock solution. Mix thoroughly using a pipette or vortex.
3. Calculation of Dye Amount
The amount of Alexa fluor 647 NHS ester required for the reaction depends on the amount of protein to be labeled. The optimal molar ratio of Alexa fluor 647 NHS ester dye to protein is approximately 10.
Example: Assuming the required marker protein is 500 μL of 2 mg/mL IgG (MW=150,000), and 1 mg of Alexa fluor 647 NHS ester is dissolved in 100 μL of DMSO, the required volume of Alexa fluor 647 NHS ester is 6.31 μL. The detailed calculation process is as follows:
1) mmol (IgG) = mg/mL (IgG) × mL (IgG) / MW (IgG) = 2 mg/mL × 0.5 mL / 150,000 mg/mmol = 6.7 × 10⁻⁶ mmol
2) mmol (Alexa fluor 647 NHS ester) = mmol (IgG) × 10 = 6.7 × 10⁻⁶ mmol × 10 = 6.7 × 10⁻⁵ mmol
3) μL (Alexa fluor 647 NHS ester) = mmol (Alexa fluor 647 NHS ester) × MW (Alexa fluor Alexa fluor 647 NHS ester)/mg/μL = 6.7 × 10⁻⁵ mmol × 942.06 mg/mmol/0.01 mg/μL = 6.31 μL (Alexa fluor 647 NHS ester)
Note: We recommend a dye/protein molar ratio of 10:1. If the concentration is too low or too high, the ratio can be adjusted to 5:1, 15:1, or 20:1.
4. Running the Coupling Reaction
1) Slowly add an appropriate amount of freshly prepared 10 mg/mL Alexa fluor 647 NHS ester to 0.5 mL of protein sample.
In the solution, gently shake to mix, then briefly centrifuge, collecting the sample at the bottom of the reaction tube. Do not mix, as this may denature and inactivate the protein sample.
2) Place the reaction tube in a dark place and gently incubate at room temperature for 60-minute intervals. 10-15 minutes, gently reverse the reaction.
5. Purify the Conjugate
The following protocol is an example of purifying dye-protein conjugates using a SepHadex G-25 column.
1) Prepare the SepHadex G-25 column according to the manufacturer's instructions.
2) Load the reaction mixture onto the top of the SepHadex G-25 column.
3) Once the sample has run below the top resin surface, immediately add PBS (pH 7.2-7.4).
4) Add more PBS (pH 7.2-7.4) to the desired sample to complete column purification. Combine the fractions containing the desired dye-protein conjugate.