| Product Description |
Product introduction：Aladdin ® SE is a kind of fluorescent dye with amino reactive activity. The SE group of these dyes can react with the amino group to produce a stable amide bond. Compared with other similar dyes on the market, aladdin ® is a new generation of fluorescent dyes with stronger stability, better water solubility and better fluorescence intensity.
Product parameters： Absmax/Em(nm)：661/679；Absmax/Em(nm)：0.51；Extinction coefficient(ε)：100,000；Optimal DOL(IgG)：4-7；
Usage:
1. Experimental materials
(1) IgG: IgG must not contain amine chemicals that can react with dyes, such as amino acids, Tris, BSA, gelatin, etc. If IgG contains such chemicals, PBS buffer with pH~7.4 should be used for pre dialysis treatment. The presence of azide compounds does not affect the labeling reaction.
(2) Anhydrous DMSO
(3) NaHCO3
(4) Sephadex gel G-25 dialysis column
(5) PBS buffer (pH~7.4)
(6) NaN3
(7) BSA
2. Marking methods and steps
(1) Prepare to label antibodies
Dilute the antibody with 0.1 M NaHCO3 solution (pH~8.3) to a final concentration of 2.5 mg/mL. If the product is pre diluted with phosphate buffer, such as PBS buffer (without amino compounds), approximately 1/10 volume of 1M NaHCO3 mother liquor can be directly added to the buffer to achieve a final NaHCO3 concentration of 0.1 M.
Note: When the protein concentration is 2.5 mg/mL, the labeling efficiency is approximately 35%. Protein concentrations below 2.5 mg/mL can also be used for labeling, but the labeling efficiency will decrease. When the protein concentration is higher than 5 mg/mL, the labeling efficiency may be higher. Due to the differences in liquid and protein purity, more precise labeling efficiency is determined by practical operating conditions. If the protein concentration is too low, it can be concentrated by ultrafiltration.
(2) Prepare dye storage solution
Preheat one tube at room temperature μ YF of Mole ® SE, add 0.1 mL of anhydrous DMSO to the tube, thoroughly vortex dissolve the dye, and prepare a dye storage solution with a concentration of 10 mM. If a trace amount of protein is used for labeling reactions, the dye needs to be diluted to a lower concentration.
Note: a The remaining dye storage solution should be stored at a low temperature of -20 ℃ for future use. If anhydrous DMSO is used to prepare dye storage solution, the dye can be stored for at least one month.
b. Dyes can also be prepared with deionized water, but due to the slow hydrolysis of dyes in water, it is best to prepare water based storage solutions for immediate use.
(3) Mark reaction steps
a. Stir or vortex the protein solution, gradually adding 15-25 drops μ L dye storage solution (10 mM), with a molar ratio of dye/protein in the range of 9:1 to 15:1. YF ® Please refer to the table above for the range of DOL (number of dyes bound to each protein molecule) for SE labeled IgG antibodies.
b. Stir the reaction at room temperature for 1 hour, and for trace labeling, shake and incubate on a shaker for 1 hour.
Note: At the same time of the binding reaction, proceed to step 2 (4) to balance the dextran gel G-25 dialysis column.
(4) Isolation of marker proteins from reaction solution
a. PBS buffer (pH~7.4) was used to balance the dextran gel G-25 dialysis column (10 mm × 300 mm).
b. Add the reaction solution from step 3 (b) to the column and elute with 1 x PBS buffer.
The first washed out chromophore is a dye protein complex.
Note: a For small-scale labeling reactions, in order to avoid excessive dilution of the product
To remove free dyes from the complex using an ultrafiltration device.
b. After the binding reaction is completed, if the dye protein complex is not separated in time, 50 can be added μ Terminate the reaction with L 1M lysine. In most cases, this operation is not necessary because the remaining unreacted dyes have been fully hydrolyzed at the end of the reaction.
3. Determine DOL
(1) Determination of protein concentration
The antibody concentration can be calculated using the following formula:
C (mg/mL)={[A280- (Amax x x Cf)]/1.4} x dilution factor;
a. C refers to the concentration of antibodies collected in the experiment;
b. Dilution factor refers to the dilution factor used in photometric measurements;
c. A280 and Amax refer to the absorbance at 280 nm and the absorbance at the absorption wavelength, respectively;
d. Cf is the correction factor, YF ® Please refer to the table above for the Cf value of SE dyes;
Note: The protein solution eluted through the column may have a high concentration when used directly for absorbance detection, so it needs to be diluted to approximately 0.1 mg/mL. Dilution factor
It is necessary to estimate from the initial amount of antibodies (such as 5 mg) and the total volume of protein elution.
(2) Estimation of DOL
DOL is calculated using the following equation:
DOL=(Amax x x Mwt x Dilution Factor)/（ ε X C)
a. Amax, dilution factor, C value has been clearly defined in 3 (1);
b. Mwt refers to the molecular weight of IgG (150000);
C. c ε It's YF ® The molar absorption coefficient of SE, refer to the table on the first page;
d. Mark YF ® The optimal DOL value for SE IgG antibodies can be found in the table on the first page. Although DOL values may fluctuate, good experimental results can also be achieved.
Matters needing attention： 1. if the labeled protein needs long-term storage, it is recommended to add 5-10 mg/ml BSA and 0.01-0.03% NaN3 to prevent protein denaturation and microbial breeding. Store at 4 ℃ away from light. If glycerol with a final concentration of 50% is added, it can be stored at -20 ℃. It can be stably stored for more than one year. 2. keep away from light during operation. The mixing speed should be appropriate to avoid bubbles. 3. when installing the chromatographic column, try to make the column body uniform, the column surface flat, and free of bubbles and cracks. 4. pay attention to adding the sample when the column top buffer is tangent to the gel plane. When eluting, add the eluent when the sample is tangent to the gel plane. 5. other factors affecting the labeling efficiency also include temperature, reaction time, pH, the amount of fluorescent dye and protein, etc., which should be controlled. 6. for your safety and health, please wear laboratory clothes and disposable gloves. Scope of application： Protein nucleic acid labeling dye |
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H2408117