VivoTag on the ASK


Overview


VivoTags® are optimized NIR labels for labeling molecules including peptides, small molecules, proteins, macromolecules, and nanoparticles. They are available as either NHS esters or maleimide reactive dyes which makes conjugation to either free amine (-Nh3) and free thiol (-SH) containing molecules possible. VivoTags have validated for both in vivo and in vitro applications. Figure 1 below shows the conjugation reaction.

vivotagfig1.jpg
Figure 1: Conjugation reaction for NHS ester (A) and maleimide (B) VivoTag dyes. VivoTag labeled conjugates have been validated in both IVIS® and FMT® imaging systems. In Figure 2 below, Nu/nu mice were orthotopically injected with the Bioluminescent cell line MDA-MB-231-luc2. An antibody-conjugate labeled with VivoTag with specifity for the tumor cells was injected and compared to a VivoTag labeled IgG with no tumor specificity.

vivotagfig2.jpg
Figure 2: Imaging VivoTag conjugates on IVIS and FMT systems. A. Bioluminescence (left) and fluorescence intensity (right) were measured using an IVIS spectrum. In both mice the biolumenscence from the tumor cell line is strong, but only the mouse on the right, injected with the tumor specific VivoTag conjugate shows fluorescence at the tumor site. B. FMT reflectance mode is used to show the mouse on the right, injected with the tumor specific VivoTag conjugate, s a strong signal in the area of the of the orthotopically injected tumor. The high signal in the liver is due the distribution kinetics of the antibody. All images shown are at 24 hours post injection of the antibody conjugate.


Products and catalog numbers


Table of VivoTag labeling agents:
Labeling agentReactive withAbs max (nm)Em max (nm)Epsilon (M-1cm-1)Molecular weight* (g/mol)Settings for in vivo imagersFilters for microscopy and flow cytometryCatalog numbers
VivoTag 645Free amines (lysines)643660~210,0001393FMT 635/655
IVIS 640/660 with spectral unmixing
Microscopy: Cy 5
Flow Cytometry: 705/70
1 mg: NEV11173
5 mg: NEV11174
VivoTag 645 MALFree thiols (cysteines)643660~210,0001418FMT 635/655
IVIS 640/660 with spectral unmixing
Microscopy: Cy 5
Flow Cytometry: 705/70
1 mg: NEV11273
5 mg: NEV11274
VivoTag 680 XLFree amines (lysines)665688~210,0001856FMT 680/700
IVIS 675/720
Microscopy: Cy 5.5
Flow Cytometry: 712/21
1 mg: NEV11119
5 mg: NEV11120
VivoTag 680 XL Labeling KitFree amines (lysines)665688~210,0001856FMT 680/700
IVIS 675/720
Microscopy: Cy 5.5
Flow Cytometry: 712/21
1 kit: NEV11118
(2 x 0.25 mg)
VivoTag 680 XL MALFree thiols (cysteines)669688~210,0001881FMT 680/700
IVIS 675/720
Microscopy: Cy 5.5
Flow Cytometry: 712/21
1 mg: NEV11219
5 mg: NEV11220
VivoTag-S 680Free amines (lysines)673691~230,0001439FMT 680/700
IVIS 675/720
Microscopy: Cy 5.5
Flow Cytometry: 712/21
1 mg: NEV10121
5 mg: NEV10122
VivoTag-S 750Free amines (lysines)750775~240,0001183FMT 750/770
IVIS 745/800
Microscopy: Cy 5.5 or Cy 7**
Flow Cytometry: 780/60
1 mg: NEV10123
5 mg: NEV10124
VivoTag-S 750 MALFree thiols (cysteines)750775~240,0001208FMT 750/770
IVIS 745/800
Microscopy: Cy 5.5 or Cy 7**
Flow Cytometry: 780/60
1 mg: NEV11223
5 mg: NEV11224
VivoTag 800Free amines (lysines)785810~200,0001464FMT 790/810
IVIS N/A
Microscopy:N/A
Flow Cytometry: N/A
1 mg: NEV11107
5 mg: NEV11108
Table 1: Available VivoTag labeling reagents.
*Molecular weight shown is for the TEA salt form
**For 750 nm probes, Cy 5.5 filters are recommended for microscopy for lower acquisition times.

Choosing the right VivoTag labeling agent

  • Dye Wavelength: Be sure to choose the proper wavelength that your instrument is capable of measuring effectively. For in vitro applications such as microscopy, dyes above 750 nm can't typically be imaged. For deep tissue measurements in vivo however, longer wavelength dyes such as VivoTag 750 or VivoTag 800 are preferred provided your in vivo imager has the proper filter sets.
  • VivoTag vs. VivoTag MAL: VivoTags are suitable for labeling free amines (typically lysine residues), while VivoTag MAL dyes are suitable for labeling free thiols (typically cysteine residues).
  • VivoTag vs. VivoTag -S: The "S" in VivoTag -S stands for "self-quenching". When a protein is labeled with multiple VivoTag-S molecules, there is a shift in the absorbance of the dye, which will ultimately reduce the signal if using the filter sets listed above. Only choose VivoTag -S if you are sure your labeling ratio of dye: protein will be 1:1. If multiple dyes are to be conjugated onto your protein, the non -"S" version should be chosen.
  • Package Size: Standalone Vivotags are available in 1 mg and 5 mg package sizes. Typically you can label 10-20 mg of antibody using 1 mg of dye, but we do recommend trying different dye: protein conjugation ratios to determine the optimal conditions for your protein of interest.


What do I need to perform a VivoTag conjugation?


You will need:
  • Appropriate VivoTag labeling reagent (see Table 1 in section above for catalog numbers)
  • Appropriate conjugation buffer
    • For NHS ester, 50 mM carbonate/biocarbonate buffer, pH 8.3-8.5
    • For maleimide, appropriate buffer (e.g. 100 mM PBS, 100 mM TRIS, or 100 mM HEPES buffer, pH 6.5-7.0)
  • Dimethylsulfoxide (DMSO)
  • Conical collection tubes
  • Appropriate filtration column
    • For NHS ester, Bio-Rad Bio-Gel P-100
    • For Maleimide, Sephadex G-25

Or:


And:

  • 0.2 µm syringe filter to filter the purified conjugate
  • Absorbance reader (UV spectrophotometer, NanoDrop spectrophotometer) to determine the degree of labeling


Protocols and calculations


General overview of conjugation protocol

vivotagfig3.jpg
Figure 3: Flow chart of conjugation protocol. 1) Starting VivoTag stock solution in DMSO varies by reagent, please click on the detailed protocols below to determine the recommended stock concentration. 2) Optimal labeling ratios will vary by protein and multiple dye: protein ratios may be have to be tested to determine the best condition for labeling your molecule of interest. 3) Labeling time varies by dye, please check with detailed protocols below for specific reaction times.

Please download more complete protocols for labeling proteins and antibodies with VivoTag:

Determining the degree of labeling

In order to determine the degree of labeling, the absorbance of the diluted VivoTag-protein conjugate is measured at both 280 nm (to determine the amount of protein present) and at the absorbance wavelength of the VivoTag dye (643 nm to 790 nm, depending on the dye-See Table 1 above). There is a small amount of crosstalk at 280 nm as the VivoTag dyes also absorb at this wavelength, so it is necessary to subtract out this background absorbance in order to precisely calculate the degree of labeling. The amount of crosstalk varies by which VivoTag dye is chosen. Table 2 lists the amount of crosstalk for each VivoTag at 280 nm.

VivoTag% Crosstalk at 280 nmCorrection factor (Cf)
VivoTag 64550.05
VivoTag 645 MAL50.05
VivoTag 680 XL160.16
VivoTag 680 XL MAL130.13
VivoTag-S 680160.16
VivoTag-S 75060.06
VivoTag-S 750 MAL50.05
VivoTag 80050.05
Table 2: Correction factors for VivoTags. The correction factor is used to calculate the background absorbance of the dye at 280 nm. See sample calculation below.

Sample Calculation
Here is an example of the calculation if you labeled of an IgG labeled with VivoTag 645. After purification, you dilute the conjugate 1:10 in PBS. You measure the A280 of the dilution to be 0.415 and the A643 of the dilution to be 0.786. The Epsilon (Ɛ) for IgG is 210,000 M-1cm-1 and the Epsilon (Ɛ) for VivoTag 645 is 210,000 M-1cm-1 (see Table 1 for all dye Epsilons).
First the A280 and the A643 need to be multiplied by the dilution factor of 10 in order to determine the stock concentration:

A280 = 0.415 x 10 = 4.15
A643 = 0.786 x 10 = 7.86


To calculate the concentration of the IgG (Pc) the correction factor needs to be multiplied by the dye absorbance and subtracted from the A280:

Pc [M] = (A280 - (A643)*Cf)/Ɛ of IgG
Pc = (4.15 - 7.86*0.05)/210,000
Pc = 4.15-0.393/210,000
Pc = 3.757/210,000 = .00001789 M or 17.89 µM IgG


To calculate the concentration of the VivoTag 645 dye:

Dc [M] = (A643)/Ɛ of VivoTag 645
Dc = 7.86/210,000
Dc = 0.0000374 M or 37.4 µM VivoTag 645


To calculate the degree of labeling:

Dc/Pc = 37.4 µM/17.89µM = 2.09


In this case, there are 2.09 VivoTag dye molecules for every molecule of IgG. Ideal dye-to-protein labeling ratios are typically between 2 and 3. It is important to note that the above equations assume a path length of 1 cm (typical path length of a cuvette). If you are using a NanoDrop spectrophotometer to measure absorbance, the path length is only 1 mm so A280 and A643 values obtained by this method would have to multiplied by an additional factor of 10. Omitting this correction factor will not alter the dye-to-protein ratio calculated, but will alter the µM of dye measured which will affect the amount of conjugate injected into your in vivo model organism.


Assay optimization in vivo


Not all proteins or antibodies make good imaging agents, independent of target specificity. Some may have too long or short of a half-life in vivo, or may excessively accumulate in non-target sites. It is a good idea to test specificity both in vitro and in vivo is possible, and to determine the in vivo biodistribution and pharmacokinetics of your VivoTag conjugate.


FAQs


General Product and Application FAQs

Q. What is the structure of VivoTag?
A. Unfortunately the structure of VivoTag is proprietary and we are unable to disclose it to customers.


Q. What is the difference between VivoTag and VivoTag -S?
A. VivoTag-S refers to a shift in absorbance that the dye experiences when conjugated on a molecule with multiple dyes. The shift in absorbance upon conjugation is about 50-60 nm lower in wavelength than the free dye alone.


Q. For VivoTag -S what does the shift in absorbance correspond to in terms of what I'll see on my instrument? Do I need to adjust any filter settings?
A. It depends on the instrument that is being used. If multiple tags in close proximity are added then the new max wavelength will be 50-60 nm toward the blue range and if it is only one VivoTag the wavelength will be the one reported on the technical data sheet.


Q. Will there be an increase or decrease in signal based on the number of VivoTag molecules that bind to my protein?
A. This will depend on the protein being used and the method of detection being used as well.


Q. What is the difference between VivoTag and VivoTag MAL?
A. The main difference between the regular dyes and the MAL dyes is that the MAL dyes are better suited for labeling thiol and cysteine containing molecules. The dye properties (abs, FL, ex. coeff, etc) are all the same.


Q. Can VivoTags be used to label cells?
A. While this is possible, we instead recommend VivoTrack™ for this application.


Q. Can I use VivoTags in humans?
A. No. VivoTags are intended for research purposes only and are not for human use.


Q. When optimizing my experiment in vivo, is there a control dye that can be used?
A. Yes, you can use Genhance™ as a control to determine non-specific uptake as this dye has no specificity for any particular target in vivo (other than vasculature). It is the acid form of the VivoTag dyes and is available as in 680 nm and 750 nm versions. Alternatively, you could conjugate a non-targeting protein or antibody to VivoTag and measure its specificity compared to your targeted molecule.


Labeling and Purification FAQs

Q. Can I reconstitute a VivoTag in an aqueous buffer?
A. Yes you technically can if your experiment requires it, but the reaction needs to be done very quickly so that the NHS group does not hydrolyze and render the dye no longer reactive.


Q. What amount of dye is a good starting point for protein or antibody labeling?
A. Typically you can label 10-20 mg of antibody or protein with 1 mg of dye. This is a good starting point, but since all molecules are unique the amounts and ratios need to be optimized.


Q. I do not want to run my conjugate over a column to purify. Can I dialyze away the free dye instead?
A. For the NHS labeling reagents, we not recommend dialysis as a way to remove free dye. For the maleimide dyes, dialysis can be used instead of a gel filtration column if desired.


Q.What mass should I use when analyzing my NHS VivoTag conjugates using mass spec?
A. This table contains the mass expected when using mass spec analysis:

VivoTagMass (X) to be added to the MW of biomolecule
VivoTag 645974.2
VivoTag 680 XL1234.2
VivoTag-S 6801019.2
VivoTag-S 750865.2
VivoTag 8001045.2

Q. Why does the table above contain values for the molecular weight that differ from the technical data sheet?
A.The VivoTags exist as triethylammonium series salts. The number of triethylammonium groups vary from bis triethylammonium to penta triethyammonium dyes. When running Mass Spec, depending on the conditions, it is possible to observe up to 5*101(MW of TEA) less than the number reported on the TD sheet. Also upon Mass Spec analyses the dye will hydrolyze giving the MW of the acid form rather than the NHSE form of the dye.


Storage and Stability FAQs

Q.How long is the VivoTag powder good for?
A. Stability of the powder will vary for each product. Please check the technical data sheet for the most accurate information about storage temperature and shelf life.


Q. How long is the reconstituted VivoTag dye good for?
A. We recommend storing the DMSO reconstituted VivoTag dye at 4 °C protected from light. Under these conditions, we recommend using within 1 week.


Q. How long is my VivoTag labeled protein or antibody good for?
A. When stored at 4 °C and protected from light, typically labeled protein is good for 4 weeks. If long term storage is needed, we recommend storing at -20 °C.


Citations


Search or browse our Citations Library for VivoTag citations.