Antibody Labelling for STORM Imaging
MATERIALS
2nd antibody stock solution
2nd antibody stock solution
METHODS
Single Labeled 2nd antibody mix
bring the total volume to 200ul before gel filtration
Mix ratio should be – Antibody : Dye ~= 1 : 20
Label ratio should be – Antibody : Dye ~= 1 : 2~5
It depends on the reaction efficiency of the Dye-NHS
Dual Labeled 2nd antibody mix
Mix ratio should be – Antibody : Dye ~= 1 : 20
Label ratio should be – Antibody : Dye ~= 1 : 2~5
It depends on the reaction efficiency of the Dye-NHS
Dual Labeled 2nd antibody mix
bring the total volume to 200ul before gel filtration
Mix ratio should be – Activator : Antibody : Reporter ~= 20 : 1 : 5
Label ratio should be - Activator : Antibody : Reporter ~= 2~5 : 1 : 0.6~1
It depends on the reaction efficiency of the Dye-NHS
The labeling ratio is calculated by concentrations of the antibody, activator dye and reporter dye. These concentrations are calculated using Beer-Lambert’s law.
For Antibody
The concentration of antibody is calculated by “A280(actual)”. A280, A405, A647 show absorbance at 280nm, 405nm and 647nm, respectively. A280(actual) shows actual absorbance of labeling antibody and A280(measured) shows a measured value by UV/Visible absorption spectrophotometer. Furthermore, the Correction Factor at 280 nm of each labeling dye (Cy2, Cy3, Alexa405, Alexa647) is represented by CF280, dye name. If you choose Alexa405 as a labeling dye partner for Alexa647, A280(actual) is calculated by the following equation.
A280(actual) = A280(measured)
– CF280, Alexa405 × A401(measured)
– CF280, Cy3 × A550(measured)
– CF280, Alexa647 × A650(measured)
CF280, Alexa405 = 0.70
CF280, Cy3 = 0.08
CF280, Alexa647 = 0.03
Concentration of Antibody = A280(actual) /εf280
εf280 = Extinction coefficient at 280 nm
εf280= 1.36 for Jackson Antibody IgG (Johnstone and Thorpe, 1988)
The concentration of labeling dye is calculated by absorbance maxima.
Measured absorbance maxima is equal to absorbance maxima as actual λmax: Aλmax(actual).
Concentration of labeling dye = Aλmax(actual) /εfλmax
εfλmax = Extinction coefficient at the wavelength of absorbance maxima
Alexa 405 → 34,000 (at 401 nm)
Cy3 → 150,000 (at 550 nm)
Alexa 647 → 239,000 (at 650 nm)
Mix ratio should be – Activator : Antibody : Reporter ~= 20 : 1 : 5
Label ratio should be - Activator : Antibody : Reporter ~= 2~5 : 1 : 0.6~1
It depends on the reaction efficiency of the Dye-NHS
The labeling ratio is calculated by concentrations of the antibody, activator dye and reporter dye. These concentrations are calculated using Beer-Lambert’s law.
For Antibody
The concentration of antibody is calculated by “A280(actual)”. A280, A405, A647 show absorbance at 280nm, 405nm and 647nm, respectively. A280(actual) shows actual absorbance of labeling antibody and A280(measured) shows a measured value by UV/Visible absorption spectrophotometer. Furthermore, the Correction Factor at 280 nm of each labeling dye (Cy2, Cy3, Alexa405, Alexa647) is represented by CF280, dye name. If you choose Alexa405 as a labeling dye partner for Alexa647, A280(actual) is calculated by the following equation.
A280(actual) = A280(measured)
– CF280, Alexa405 × A401(measured)
– CF280, Cy3 × A550(measured)
– CF280, Alexa647 × A650(measured)
CF280, Alexa405 = 0.70
CF280, Cy3 = 0.08
CF280, Alexa647 = 0.03
Concentration of Antibody = A280(actual) /εf280
εf280 = Extinction coefficient at 280 nm
εf280= 1.36 for Jackson Antibody IgG (Johnstone and Thorpe, 1988)
The concentration of labeling dye is calculated by absorbance maxima.
Measured absorbance maxima is equal to absorbance maxima as actual λmax: Aλmax(actual).
Concentration of labeling dye = Aλmax(actual) /εfλmax
εfλmax = Extinction coefficient at the wavelength of absorbance maxima
Alexa 405 → 34,000 (at 401 nm)
Cy3 → 150,000 (at 550 nm)
Alexa 647 → 239,000 (at 650 nm)