YO425SE (YO425 succinimidyl ester) - 90%, high purity

Item Number

Y598204

• Specifications & Purity: 90%
• Storage Temp: Protected from light,Store at -20°C
• Shipped In: Ice chest + Ice pads

Product Description

Uelandy provides a variety of cost-effective amino reactive dyes for labeling proteins and other amine containing compounds. YO dyeSE can react with amino groups to form stable amide bonds.

Product parameters：

Absmax/Em(nm)：429/475；Absmax/Em(nm)：0.17；Extinction coefficient(ε)：45000；Optimal DOL(IgG)：2-3

Spectrogram：

( Dissolved in DMSO )  

Instruction： 

1. Experimental materials
(1) Protein solutions must not contain amine chemicals that can react with dyes, such as amino acids, Tris, BSA, gelatin, etc. If the protein contains such chemicals, PBS buffer with pH~7.4 should be used for pre dialysis treatment. The presence of low concentration (<3 mM) sodium azide does not cause interference or labeling reactions.
(2) Solution A: PBS buffer (pH 7.4).
(3) Solution B: 0.2 M NaHCO3 solution, adjust the pH to 9.0 with 2 M NaOH.
(4) Solution C: Add 1 part of solution B to 20 parts of solution A to obtain a labeled buffer with a pH of 8.3. Store in a sealed bottle, this solution can remain stable for a long time.
(5) Sephadex G-25 gel filtration column.
2. Marking methods and steps
(1) Dissolve 1-5 mg of protein in 1 mL of solution C.
(2) Dissolve 1 mg of dye in 50-200 µ L anhydrous, amine free DMSO, shake thoroughly and mix well.
(3) Add an appropriate volume of dye to the protein solution, so that the molar ratio of dye to protein is 3:1. Due to the different reactivity of proteins and labeling reagents, changes may occur. This may require optimizing the ratio of dyes to proteins used in the reaction to obtain the required DOL.
(4) Incubate at room temperature for 1 hour. For YO565 SE, it is recommended to incubate for 18 hours to complete the labeling reaction.
(5) Balance the dextran gel G-25 dialysis column with solution A.
(6) Add the reaction solution in step (4) to the column and elute with solution A. The first washed out chromophore is a dye protein complex.
Note: To prevent denaturation of the conjugate after elution, bovine serum albumin (BSA) or other stabilizers can be added.
3. DOL calculation: DOL=c (dyne)/c (protein)=(Amax)/ ε Max/(Apro)/ ε Pro=(Amax)· ε Pro)/[(A280- Amax · Cf)· ε Max]
A. a ε Pro is the molar absorbance coefficient of the protein;
b. A280 and Amax refer to the absorbance at 280 nm and the absorbance at the maximum absorption wavelength, respectively;
c. Cf is the correction factor, refer to the table above;
D ε Max is the molar absorption coefficient of the dye.

Matters needing attention：

1. the excitation and emission wavelengths of fluorescent dyes will be affected by solvents. If different solvents are used, the excitation and emission wavelengths may change. 

2. if the labeled protein needs to be stored, NaN3 with a final concentration of 2 mm can be added as a preservative and stored at 4 ℃ away from light. For long-term storage, please divide the solution into small portions and freeze at -20 ℃, avoid repeated freezing and thawing, and protect the dye conjugate from light as much as possible. 

3. avoid light and bubbles during operation. 

4. when installing the chromatographic column, try to make the column body uniform, the column surface flat, and free of bubbles and cracks. 

5. 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. 

6. other factors affecting the labeling efficiency also include temperature, reaction time, pH, the amount of fluorescent dye and protein, etc., which should be controlled. 

7. for your safety and health, please wear laboratory clothes and disposable gloves.

Scope of application：

Protein nucleic acid labeling dye