Properties of Super Fluor 555 activated ester
Activated ester molecular weight: 1250
Maximum absorption wavelength (nm): 555
Molar absorption coefficient (M-1cm-1): 150000
Maximum emission wavelength (nm): 565
A280nm / Amax: 8%
Reference scheme for Super Fluor labeled antibodies
1) Protein dissolution: Dissolve with 0.1 M NaHCO3 to a final concentration of 2mg/mL
Add 1ml of deionized water to an 84mg NaHCO3 bottle to make a 1M sodium bicarbonate solution. Shake or pipette until completely dissolved. The pH of the solution is about 8.3, and it can be stored at 2-6°C for 2 weeks; if the antibody is a solution, dilute the antibody to 2mg/mL, then add 1/10 volume of the above sodium bicarbonate solution; if the antibody is a lyophilized powder, add 1M After the sodium bicarbonate solution is diluted with 10 times deionized water to 0.1M, use it to dissolve the antibody into a 2mg/mL solution;
2) Dye dissolution: After the activated Super Flour SE dye solid powder is slowly warmed to room temperature in a desiccator from the refrigerator, the dye preparation concentration is 1 mg/mL with anhydrous DMSO.
3) Labeling: Slowly add an appropriate amount of dye solution to 1 mL of protein solution, (protein: dye molar ratio = 1:20-50; the mass ratio can be optimized within the following range of 10-200ug Super Flour SE per mg of IgG antibody). At the same time, slowly stir for 1 hour at room temperature in the dark. It is also possible to directly add the protein solution directly to the reagent bottle of the solid powder of the dye, and at the same time, slowly stir for 1 hour at room temperature in the dark.
4) Purification: After purifying the product by dialysis or Sephadex, the product is freeze-dried into powder or stored in 0.01 M PBS/2mM sodium azide solution at -20°C and protected from light.
5) Storage: Store the labeled protein at 2～6℃, away from light. If the concentration of the purified protein conjugate is less than 1 mg/mL, add BSA or other stable proteins at 1-10 mg/ml. In the presence of 2mM sodium azide, it can be stored for several months at 2-6 degrees. For longer storage, you need to store at -20°C after packaging. Avoid repeated freezing and thawing, and store in the dark.
Calculate the mark ratio F/P value
For most antibodies such as IgG, the molar absorption coefficient is 203000, and the F/P value between 4-9 is the most appropriate.
Super Flour 555 labeled protein F/P value calculation
1) Dilute quantitatively a few purified coupled proteins with PBS with precise multiples, and measure the light absorption at 280nm and 555nm in a 1cm cuvette;
2) Calculate the protein concentration in the sample: protein molar concentration=(A280-(A555×0.08))×dilution multiple/203000
3) Calculate the labeling ratio: moles of dye bound per mole of protein = A555 × dilution factor / (150000 × protein molar concentration)
Reactive dye stability and storage
Please note: the unopened powder can be stored for 12 months in the dark and dry at -20℃. The aqueous solution can not be stored when used. Any dissolved dye is best used immediately; anhydrous DMSO solution is stored at -20°C for up to 2 weeks.
B2323446