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HeLa cells were incubated with (Tubulin+) or without (Tubulin-) mouse anti-tubulin and biotin goat anti-mouse IgG followed by FITC-Xtra streptavidin conjugate (Green, Left) or FITC-streptavidin conjugate (Green, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17530).
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Ex/Em (nm)496/524
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Category Superior Labeling Dyes
iFluor Dyes and Kits
Related Amine-Reactive Probes
Labeling via Amino Groups
Although FITC is still the most popular fluorescent labeling dye for preparing green fluorescent bioconjugates, there are certain limitations with FITC, such as severe photobleaching for microscope imaging and pH-sensitive fluorescence. Protein conjugates prepared with FITC-xtra are far superior compared to the corresponding FITC conjugates. FITC-xtra conjugates are significantly brighter than FITC conjugates and are much more photostable. Additionally, the fluorescence of FITC-xtra is not affected by pH (4-10). This pH insensitivity is a major improvement over FITC, which emits its maximum fluorescence only at pH above 9. FITC-xtra has spectral properties almost identical FITC. In addition, FITC-xtra give much higher conjugation yield under mild conjugation conditions than FITC. Like 5-FITC, FITC-xtra antibody conjugates have excitation ideally suited to the 488 nm laser line, making them alternatives to the corresponding FITC-labeled antibody conjugates. Under the same conditions tested, FITC-xtra antibody conjugates give much higher signal/background ratios than the corresponding FITC-labeled conjugates.

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of FITC-xtra to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

Molarity calculator

Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
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This protocol only provides a guideline, and should be modified according to your specific needs.
  1. Prepare protein stock solution (Solution A):
    Mix 100 µL of a reaction buffer (e.g., 1 M sodium carbonate solution or 1 M phosphate buffer with pH ~9.0) with 900 µL of the target protein solution (e.g. antibody, protein concentration >2 mg/ml if possible) to give 1 mL protein labeling stock solution.

    Note 1: The pH of the protein solution (Solution A) should be 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer.
    Note 2: The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation.
    Note 3: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results.
    Note 4: The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.
  2. Prepare dye stock solution (Solution B):
    Add anhydrous DMSO into the vial of FITC-xtra to make a 10-20 mM stock solution. Mix well by pipetting or vortex.

    Note: Prepare the dye stock solution (Solution B) before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce the dye activity. Solution B can be stored in freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.
  3. Prepare the desired protein conjugate:
    Note: Each protein requires distinct dye/protein ratio, which also depends on the properties of dyes. Over labeling of a protein could detrimentally affects its binding affinity while the protein conjugates of low dye/protein ratio gives reduced sensitivity. We recommend you experimentally determine the best dye/protein ratio by repeating Steps 4 and 5 using a serial different amount of labeling dye solutions. In general 4-6 dyes/protein are recommended for most of dye-protein conjugates.
    1. Use 10:1 molar ratio of Solution B (dye)/Solution A (protein) as the starting point: Add 5 µl of the dye stock solution (Solution B, assuming the dye stock solution is 10 mM) into the vial of the protein solution (95 µl of Solution A) with effective shaking. The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD.
      Note: The concentration of the DMSO in the protein solution should be <10%.
    2. Run conjugation reaction. Add the appropriate amount of dye stock solution (Solution B) into the vial of the protein solution (Solution A) with effective shaking. Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes.
    3. Repeat #3.2 with the molar ratios of Solution B/Solution A at 5:1; 15:1 and 20:1 respectively if desired.
    4. Purify the desired conjugates using premade spin columns or other techniques.
    5. Calculate the dye/protein ratio (DOS) for the above 4 conjugates (see below).

References & Citations

Role of reactive oxygen species-mediated MAPK and NF-$\kappa$B activation in polygonatum cyrtonema lectin-induced apoptosis and autophagy in human lung adenocarcinoma A549 cells
Authors: Tao Liu, Lei Wu, Di Wang, Haiyang Wang, Jinwu Chen, Chunlan Yang, Jinku Bao, Chuanfang Wu
Journal: Journal of Biochemistry (2016): mvw040

Determination of sodium benzoate in food products by fluorescence polarization immunoassay
Authors: Linlin Ren, Meng Meng, Peng Wang, Zhihuan Xu, Sergei A Eremin, Junhong Zhao, Yongmei Yin, Rimo Xi
Journal: Talanta (2014): 136--143

Complexes containing cationic and anionic pH-sensitive liposomes: comparative study of factors influencing plasmid DNA gene delivery to tumors
Authors: Yan Chen, Ji Sun, Ying Lu, Chun Tao, Jingbin Huang, He Zhang, Yuan Yu, Hao Zou, Jing Gao, Yanqiang Zhong
Journal: International journal of nanomedicine (2013): 1573

Swimming behavior of the monotrichous bacterium Pseudomonas fluorescens SBW25
Authors: Liyan Ping, Jan Birkenbeil, Shamci Monajembashi
Journal: FEMS microbiology ecology (2013): 36--44