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You can perform the reaction in DCM or DMSO by adding 1 - 1.2 eq. of diisopropyl ethyl amine (Hünigs base) and a 1.3 fold molar excess of the dye. Incubate at room temperature for 1 - 2 hours.
Please note that if you need to treat your peptide with reducing agents like TCEP, DTT etc. to ensure the presence of free thiol groups, great care must be taken by removing any excess of these reagents before the labeling reaction as they consume dye maleimide themselves.
Please follow the recommendations in our labeling procedures for amine reactive probes under Support, "User Guides & Protocols". The recommended pH for antibody labeling is 8.3. Due to the high reactivity and purity of the ATTO NHS-esters it is very important - and this holds in particular for ATTO 647N - that you only use a 3 - 5 fold molar excess of the dye -NHS-ester to antibody. This will generally result in an antibody-dye conjugate with a degree of labeling (DOL) in the range 2.5 - 4.
A 20-fold molar excess of dye to protein will almost certainly overload your antibody resulting in non-specific binding and antibody precipitation.
For a fluorescein based assay we would recommend ATTO 488 as the FRET-donor. For the acceptor it is important to know your detection channel and possible filter-sets. ATTO 550, ATTO 590 and ATTO 647N are often used in combination with ATTO 488 in FRET applications. For a more red-shifted assay ATTO 550, ATTO 565, and ATTO 590 can be used as the donor with ATTO 647N as the acceptor. More information on ATTO-FRET pairs and a table of R(0) values for all ATTO-dye combinations can be found under Support "R(0)-Values (FRET)" and under Worth Knowing, "Förster Resonance Energy Transfer (FRET)" respectively.
You will also find self-quenching for ATTO dyes such as the ATTO 488 and ATTO 550, although for these two dyes the effect will be much more pronounced for ATTO 550.
Often, concentration- or self-quenching of fluorescence is a consequence of dye-aggregation. This is usually easy to detect in the absorption spectrum of an aqueous dye solution by the appearance of an additional band at shorter wavelength. Thus, in the case of ATTO 550, you will detect aggregation in the absorption spectrum at concentrations of 10-5 - 10-4 mol / l. For ATTO 488, however, the absorption spectrum does not change even at much higher dye-concentrations. Nevertheless, fluorescence can also be quenched in a concentration-dependent manner by interaction of the dye molecules in the excited state.
The molecular structure of the linker between the dye molecule and the reactive moiety can be found under "Dye Derivatives" in the Support section. The structure of the dye is available under the product information of "ATTO 680". Please note that all linker are flexible and therefore the length/distance of the reactive moiety and the chromophore strongly varies with conformational changes of the molecule.
The two given molecular weights have the following meaning: MW of 828 is the molecular weight of ATTO 680-NHS including the counter-ion. M+ of 623 is the molecular weight of the dye-cation found in LC-MS-analysis.
Without knowing the exact experimental setup, a possible explanation for the observation could be a too concentrated dye solution. In such a case and a 90 ° geometry you will find reabsorption of the emission resulting in cutting off the shorter wavelengths of the emission and an emission maximum shifted to longer wavelength. To minimize this phenomenon we recommend to use a dye solution with absorbance values below 0.05. For additional information visit "Determination of Fluorescence Quantum Yield" under "Worth Knowing".
If stored in its solid form at -20 °C the product is stable for at least 3 years. However, if dissolved in DMSO or DMF the shelf-life is significantly reduced depending on the quality of the solvent used.
You can download the absorbance and emission spectra of all ATTO dyes (carboxy derivative) by navigating to the tab "Spectra" within each product description. Please click on the appropriate .txt-file to save the absorbance and emission spectrum of the dye.
ATTO 490LS was dissolved in PBS pH 7.4 at a concentration of about 10-6 M for the emission (right angle, 1 cm cell) and 2 x 10-6 M for the absorbance (1 cm cell) measurement.
Dilution with acidified ethanol (0.1 vol.-% trifluoroacetic acid) is only necessary if you want to determine the concentration of your stock-solution in DMSO (or recommended solvent). By doing so you avoid dye aggregation and in some cases (ATTO 565 and ATTO 590) formation of a colorless spiro-lacton.
ATTO 550 NHS-ester is isolated as its perchlorate salt. In most cases for ΔM (MW - M+) = 100 the counter ion is a perchlorate anion.
ATTO 647 is not suitable for oligonucleotide labeling. For oligonucleotide synthesis we recommend ATTO 647N or the recently introduced label ATTO 643.
ATTO 647N is stable in TFA at room temperature for several hours. Cleaving various protecting groups using TFA or mixtures of TFA with e.g. acetonitrile, at our side went without any problems, even at elevated temperatures.
The color you are observing is to expect due to the high concentration of the stock solution. If you dilute the dye solution it will become fluorescent and you will see the yellow-green emission. In highly concentrated stock solution the emission is quenched.
ATTO 488 was dissolved in aqueous PBS at pH 7.4. The curves shown are absorbance (not excitation) and emission spectra recorded at 22 °C. This is the case for all spectra presented on our website. When there was a tendency for dye aggregation, the solution was diluted sufficiently to exhibit the absorbance spectrum of the monomers.
Your vendor IDT is right. The fluorescence of most dyes is quenched by guanine or guanosine (G). However, as you mentioned correctly, some dyes are quenched more than others. For instance oxazine dyes like ATTO 655, ATTO 680 and ATTO 700, which are good electron acceptors and therefore easy to reduce, are quenched very efficiently by G. Rhodamines such as ATTO 565 are not as easily reduced, thus are much less susceptible to this type of fluorescence quenching.
No, that is not to be expected. All ATTO NHS-esters are stable for several days at room temperature. That is also the reason why we do not ship them in a "Cold Box" or using dry ice. It is not necessary. However, for long-term storage we recommend to freeze the product at -20°C. The product is then stable for at least three years.
We checked this issue with the supplier of the sample vials and received the information that the use of chloroform is safe. 1 mg of ATTO 633 DOPE will dissolve in 1 ml of chloroform. However, we recommend that the dissolved lipid stock solution be transferred to a glass vial.
Please note that we generally recommend using a mixture of chloroform / methanol 8:2 to dissolve the ATTO dye-labeled lipids, especially for very hydrophilic dyes such as ATTO 488, ATTO 532, ATTO 542, etc.
In the case of ATTO 647N carboxy, a slight hypsochromic shift of the absorption maximum from 646 nm to 644 nm can be observed in a dye solution in PBS, at a concentration of 2 μM after a bleaching of 25 %. This phenomenon can be observed in many dyes that undergo photochemical reactions. In case of ATTO 647N this effect is comparably small.
The fluorescence quantum yield as well as the fluorescence lifetime of all ATTO dyes has been measured in aqueous buffer (PBS) at pH 7,4 and 22°C.
When there was a tendency for dye aggregation, the solution was diluted sufficiently to exhibit the absorbance spectrum of the monomers undisturbed by dimers.
The structure of the ATTO 647N carboxy derivative is given on the product data sheet and can be downloaded from our website at the actual product site. The structure of the azide linker as well as the structures of all ATTO dye modifications can be found by following the link below:
or in our catalogue at p22-24.
Yes, the fluorescence quantum yield as well as the fluorescence life time has been determined in PBS pH 7,4 at 22 °C. Please note, that all values given are valid for the carboxy derivative only. The quantum yield and decay time may be reduced dramatically for dye maleimides or in particular dye tetrazines. However, this is of no avail: As soon as the dye is coupled to a substrate/target the fluorescence is restituted.
ATTO 647 is a carbopyronin dye. As such the chromophoric system carries an electrical charge of +1. However, in addition ATTO 647 contains a sulphonic acid group and one carboxylic acid group (ATTO 647 carboxy). Now we need to distinguish whether you look at the charge before or after coupling the dye via its various modifications to a target molecule. The free dye (carboxy derivative) in aqueous solution and neutral pH would carry a net electrical charge of - 1 - reflecting the situation of a deptotonated sulphonic acid (-SO3-) and carboxylic acid (COO-) function. Lowering the pH to 2 and below would result in protonated acid functionalities (-SO3H and -COOH) and the electrical charge of ATTO 647 would change to +1.
After coupling to a substrate, the carboxylic acid function will be converted into an amide (-CONH-). Again, assuming a physiological pH the net electrical charge of the dye moiety in the conjugate will be neutral (0) - given by a positively charged chromophore and a negatively charges sulphonate group (-SO3-). This would change if you drop the pH to values below 2 when the sulphonate group is mostly protonated (-SO3H). The dye moiety would then be positively charged.
The structure of the ATTO 488 carboxy and ATTO 647 carboxy derivative is given on the product data sheet that can be downloaded from our website at the actual product site for each dye under "Documents". The product sheet also provides the molecular weight (MW) of all available dye derivative as well as the M+ value for mass spec analysis. The structure of the maleimide linker as well as the structures of all ATTO dye modifications can be found by following the link below:
or in our catalogue at p22-24.
For the information of the mass that needs to be added for each ATTO maleimide after coupling the dye to a target molecule please visit our procedures at:
www.atto-tec.com/images/ATTO/Procedures/Mal.pdf
You will find the data in Table 1 in the column Δm.
The fluorescence quantum yield (QY) of all ATTO dyes was determined in PBS pH 7.4 at 22 °C.
However, we would not recommend using the ATTO 425 maleimide or any dye maleimide as a reference for comparing QY. The maleimide moiety significantly quenches the emission of the fluorophore. Therefore, we suggest using ATTO 425carboxy as a QY reference for your oligonucleotide conjugate.
An alternative could be to take an aliquot of the dye-maleimide sock solution and add, e.g. acetylcysteine (1-1.1eq) or any other thiol containing reagent to convert the maleimide to the corresponding conjugate. By doing so, the maleimide is transformed to a succinimide derivative and the maleimide quenching is no longer given.
First of all, the degree of labeling (DOL), determined by the absorption spectrum of the dye conjugate, represents the average number of fluorophores for each streptavidin molecule. Consequently, a DOL of 3 does not mean every streptavidin molecule carries 3 fluorophores. There are streptavidin molecules that can be labeled with 1, 2, 3, 4, 5, 6, … or not be labeled at all.
The actual DOL-value can be different for different ATTO dyes and may also slightly vary between batches. You will find the DOL of your dye LOT on the certificate of analysis of your product that can be downloaded from our website at
www.atto-tec.com/shop_content.php
by selecting the dye, the modification streptavidin and by typing in the LOT-number of the product.