Use freshly prepared extracts for protein labeling is highly recommended. Use of extracts from frozen tissues or cell lines with low viability, or old protein extracts may give inadequate results.
For successful labeling, the extract should be clear. If small particles are observed, it is recommended to perform a rapid centrifugation just before the labeling procedure.
Excess of Cy3/Cy5 dye is eliminated by a rapid and easy method using SigmaSpin columns (Product No. S0185). Other methods for eliminating the excess of dyes, such as PD10 columns or dialysis can be used.
The dye to protein ratio (D/P ratio) should be >2. If this ratio is not achieved, a new sample should be labeled.
In cases where 1mg of nuclear protein extract is not obtained, lower amounts can be labeled. However, the ratio of dye to protein in the labeling mix should be kept the same. The Cy3/Cy5 dyes can be dissolved in 50-100 µl Carbonate-Bicarbonate Buffer pH 9.5-9.6 and used for the labeling procedure. For example if 400 µg of nuclear extract is obtained then 20 µl of Cy3 or Cy5 can be used (assuming the dyes were dissolved in a total volume of 50 µL of buffer.
- Use 1 mL of extract (1 mg/mL) for labeling with Cy3 or Cy5. Add 1mL of extract solution to the dye vial. Cap the vial and mix thoroughly. Care should be taken to prevent foaming of the protein solution.
- Incubate the reaction at room temperature for 30 minutes, mixing the solution every 10 minutes.
- Remove the free Cy3/Cy5 from the labeled sample by applying on SigmaSpin columns (Product No. S0185) as follows:
- Loosen the cap of the column by half a turn and then snap off the bottom closure.
- Place the column in a microcentrifuge tube and centrifuge for 2 minutes at 750 x g.
- Discard the eluate.
- Place the column in a new collection tube.
- Pipette 150 µL of the labeled protein sample solution directly onto the center of the SigmaSpin column. The remaining labeled extract can be stored at -70 °C.
- Centrifuge for 4 minutes at 750 x g.
- Discard the column and retain the eluate. This is the labeled protein sample. Protect it from prolonged exposure to light.
- Determine the protein concentration by Bradford method.
- Store the labeled protein at 2–8 °C. The sample may be frozen in case it is not possible to proceed immediately to the next step.
Determination of dye to protein molar ratio (D/P ratio)
- Measure the Cy3 and Cy5 absorbance at 552 nm and 650 nm, respectively. Read the absorbance of the dyes at their exact absorbance wavelengths maxima. Use Buffer A as the blank.
- Calculate the molar concentration of Cy3 and Cy5 taking into account the following:
• For a non-homogenous extract that contains a mixture of proteins, the protein molecular weight should be taken
as 60 kDa.
• The µmolar extinction coefficient (εmM) of Cy3 and Cy5 are:
Cy3: εmM(552 nm) = 0.15 µM-1cm-1
Cy5: εmM(650 nm) = 0.25 µM-1cm-1
Cy3 concentration (µM) = A552/0.15
Cy5 concentration (µM) = A650/0.25
Y (mg/mL) = protein concentration after labeling (see Step IIA-4)
Protein (µM) concentration = Y (mg/mL) /60,000 ] x 106
||Cy3 or Cy5 concentration
Protein concentration of sample