Fluorescent Dye

IgG Fluorescent Labeling with AF488 NHS Ester and Anti-IgG Solid-Phase Peptide Library Screening
(1) Dissolve lyophilized IgG at a concentration of 5 g/L in 50 mM sodium phosphate, 20 mM sodium chloride, pH 8.3.
(2) Dissolve 1 mg AF488 NHS ester in 100 µL extra dry DMF, then add to 1 mL of solution from step (1). Rotate and incubate at room temperature for 1 h.
(3) Collect the sample using an Amicon Ultra 0.5-mL centrifugal filter device with a 3-kDa MWCO membrane.
(4) Wash the hexamer or tetramer deprotected library three times with 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.4 (PBS) using 5× the resin volume for equilibration.
(5) Dilute IgG-AF488 to a final concentration of 1.3 mg/mL with 50 mM sodium phosphate, 150 mM sodium chloride, 0.2% Tween-20, pH 7.4.
(6) Incubate mixtures from (4) and (5) at 2-8°C overnight.
(7) Wash the resin beads with 50 mM sodium phosphate, 150 mM sodium chloride, 0.1% Tween-20, pH 7.4 (PBS-T).
(8) Deposit one bead per well in a 96-well plate with 40 µL PBS-T, then image under a fluorescence microscope at 10× magnification. Perform Alexa Fluor 488 fluorescence measurement and screening using 480 nm excitation and 510 nm emission intensity as the threshold.

AF488 NHS Ester Stock Solution1
Prepare 20 mM AF488 NHS ester in DMF.
Note: The AF488 NHS ester stock solution should be aliquoted and stored at -20°C protected from light.
Key points about AF488 NHS ester:
• It produces bright green fluorescence under 488 nm excitation with excellent photostability.
• Commonly used for labeling antibodies, proteins, and other biomolecules.
• The NHS ester group reacts with primary amines to form stable amide bonds.

Labeling Reactions [1]
Labeling reactions were conducted by combining the appropriate volumes of 20 mM AF488/Alexa Fluor 488 NHS Ester, 10 mM DIEA, amine, and solvent. Reactions were incubated in the dark overnight (16–24 hrs) unless otherwise indicated. After incubation, reactions were diluted into the separation buffer at a 5 : 100 ratio unless otherwise indicated.

---

Capillary Electrophoresis [1]
Capillary electrophoresis (CE) separations were conducted on a Beckman Coulter P/ACE MDQ capillary electrophoresis system equipped with 488 nm laser-induced fluorescence (LIF) detection. The capillary was rinsed using pressure with the separation buffer for two (2) minutes, and then the sample was injected (pressure) for 5 seconds. Separations were conducted at 15 kV for 15 minutes. After separation, the capillary was rinsed using pressure with pure water for five minutes. Capillary conditioning using 1 M NaOH was conducted with a 5-minute rinse as needed. Separation buffers tested included 10 mM carbonate (pH 10) and 10 mM carbonate with 12 mM SDS (pH 10).

---

Fluorescent Labeling of IgG and CHO-S HCPs [2]
HCPs and IgG were fluorescently labeled with Alexa Fluor NHS esters as guided by the manufacturer’s recommendations [31]. Briefly, wild-type CHO-S clarified harvest was concentrated to 2.3 g protein/L (≈6-fold) and diafiltered into 50 mM sodium phosphate, 20 mM sodium chloride, pH 8.3 using Macrosep Advance 3-kDa MWCO Centrifugal Devices. Lyophilized polyclonal human IgG (Athens Research) was dissolved in 50 mM sodium phosphate, 20 mM NaCl, pH 8.3 at a concentration of 5 g/L. 1 mg Alexa Fluor 596 NHS Ester (AF596) or Alexa Fluor 546 NHS Ester (AF546) for the HCP solution (based on the instrument to be used for fluorescence screening) and 1 mg Alexa Fluor 488 NHS Ester (AF488) for the IgG solution were each dissolved in 100 µL extra dry DMF, which was immediately combined with 1 mL of the diafiltered harvest (HCP-AF596 or HCP-AF546) or IgG (IgG-AF488) and incubated at room temperature on a rotator for 1 h. After incubation, the samples were diafiltered into 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.4 using Amicon Ultra 0.5-mL Centrifugal Filter Unit with 3-kDa MWCO filters to remove unreacted Alexa Fluor dye.

---

Fluorescence Screening of Solid Phase Peptide Libraries Against IgG and CHO-S HCPs [2]
The hexameric or tetrameric deprotected libraries were washed three times in 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.4 (PBS) at 5× the settled resin volume to equilibrate. HCP-AF596 or HCP-AF546 and IgG-AF488 were diluted in 50 mM sodium phosphate, 150 mM sodium chloride, 0.2% Tween, pH 7.4 for a final concentration of ≈1.3 mg/mL IgG-AF488, ≈0.58 mg/mL HCP-AF546 or HCP-AF596, 50 mM sodium phosphate, 150 mM sodium chloride, 0.1% Tween 20, and mixed with the washed, equilibrated libraries and incubated at 2–8°C overnight. After incubation, the excess protein solution was removed and the resin beads were washed with 50 mM sodium phosphate, 150 mM sodium chloride, 0.1% Tween 20, pH 7.4 (PBS-T). For manual fluorescence screening, the resin was deposited 1 bead per well in a 96-well plate in 40 µL PBS-T, then imaged at 10× magnification using fluorescence microscopy using a Leica DMi8 inverted microscope with a Hamamatsu C13440 digital camera and equipped with a Lumencor spectra light engine. Lead candidate beads were selected based on the highest observed emission intensity at 630 nm with excitation at 560 nm for Alexa Fluor 594 fluorescence measurement after thresholding based on 510 nm emission intensity at 480 nm excitation for Alexa Fluor 488 NHS Ester fluorescence measurement.

To increase throughput, a ClonePix 2 colony picker was used for fluorescent imaging and higher throughput sorting of HCP positive and IgG negative beads. The colony picker was identified as a possible option to increase throughput due to (1) its ability to quickly image and quantify intensity of large quantities of beads, and (2) the size range of the ChemMatrix beads, which are similar to colonies traditionally picked using the ClonePix 2 instrument. After library incubation with fluorescently tagged proteins and washed as described above, they were suspended in a semi-solid matrix to accommodate imaging and picking. The semi-solid matrix was prepared from two parts Molecular Devices CloneMatrix and three parts 83.3 mM sodium phosphate, 250 mM NaCl, 0.17% Tween 20 to generate a matrix with buffer conditions similar to the protein binding condition used. Approximately 5 to 10 µL settled volume of incubated library was gently incorporated into the matrix solution, then evenly aliquoted across a 6-well plate to obtain a target bead density of ≈100–200 beads per well. The plates were then incubated at 37 °C for 2–18 h to cure the matrix. Plates were imaged using the ClonePix FITC (800 ms exposure, 128 LED intensity) and Rhod (500 ms, 128 LED intensity) laser lines to monitor the presence of Alexa Fluor 488 NHS Ester and Alexa Fluor 546, respectively. Due to slight autofluorescence of the ChemMatrix beads under the FITC filter, bead location (i.e., ClonePix 2 run “Prime Configuration”) was assigned based on fluorescence intensity from the FITC filter. Beads were picked for further processing based on the following characteristics using the ClonePix 2: FITC interior mean intensity < 2500, Rhod interior mean intensity > 100, and 0.05–0.25 mm radius. Picking was performed in suspension mode, with 20 µL aspiration volume to pick up the bead, and a 60 µL expel volume, where excess volume above the aspirated liquid was water.

[1]. Amine Analysis Using AlexaFluor 488 Succinimidyl Ester and Capillary Electrophoresis with Laser-Induced Fluorescence. J Anal Methods Chem. 2015;2015:368362.

[2]. Targeted Capture of Chinese Hamster Ovary Host Cell Proteins: Peptide Ligand Discovery. Int J Mol Sci. 2019 Apr 8;20(7):1729.

[3]. Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides. Nucleosides Nucleotides Nucleic Acids. 2008 Sep;27(9):1072-83.

Fluorescent probes enable the detection of substances that are not normally fluorescent via highly sensitive laser-induced fluorescence. While most organic amines are non-fluorescent, they hold significant analytical value in agricultural and food science, biomedical applications, and biological warfare detection. Alexa Fluor 488 N-hydroxysuccinimide ester (AF488 NHS-ester) is an amine-specific fluorescent probe. In this paper, we demonstrate the low detection limit of this probe for long-chain (C9 to C18) primary amines and optimize the derivatization reaction of AF488 with these amines. The results show that the reaction exhibits the same efficiency in all studied solvents (dimethyl sulfoxide, ethanol, and N,N-dimethylformamide). Although an organic base (N,N-diisopropylethylamine) is required for efficient reaction of AF488 NHS-ester with organic amines possessing long hydrophobic chains, high concentrations (>5 mM) of the organic base lead to increased levels of ethylamine and propylamine in blank samples. The optimal incubation time is 12 hours or more at room temperature. We present a preliminary capillary electrophoresis separation analysis method using a simple micellar electrokinetic chromatography (MEKC) buffer consisting of 12 mM sodium dodecyl sulfate (SDS) and 5 mM carbonate at pH 10. Under optimized labeling and separation conditions, the detection limit is 5–17 nM. The method presented here adds a new option to a library of fluorescent probes that can be used for highly sensitive analysis of small organic molecules. [1]

---

The increasing popularity of the Quality by Design (QbD) concept in biomanufacturing requires a detailed and quantitative understanding of the impurity profile and its impact on product safety and efficacy. Of particular importance is the determination of residual levels of host cell proteins (HCPs) secreted by recombinant cells used in production, which are secreted along with the target product. Although often considered as a single impurity, host cell proteins (HCPs) comprise a variety of types with varying abundance, size, function, and composition. Removing these impurities is a complex problem due to variability between cell lines, products, and batches. Improvements in HCP monitoring techniques based on proteomics have enabled the identification of a subset of problematic HCPs that are difficult to remove during product capture and purification, even at extremely low concentrations, impacting product stability and safety. This paper describes the development of synthetic peptide ligands capable of capturing a variety of Chinese hamster ovary (CHO) HCPs and achieving advanced mixed-mode binding with multiple peptides. We screened a solid-phase peptide library to identify and characterize peptides that capture CHO HCPs and bind minimally to human IgG (used as a model product in this study). The study found that tetrameric and hexammeric ligands with multipolar or hydrophobic/positively charged amino acid compositions were most effective. The tetrameric multipolar ligands exhibited the highest targeting binding rate (HCP clearance rate to IgG loss rate), more than twice that of commercially available mixed-mode and anion-exchange resins used for IgG purification. All tested peptide resins showed preferential binding to HCPs over IgG, indicating potential applications in flow-through or weak partitioning mode chromatography. [2]

---

We present an efficient DNA synthesis method that completely replaces four deoxynucleoside triphosphate (dNTP) substrates with fluorescently labeled nucleotides. A spectrally separable fluorescent dye suitable for single-molecule fluorescence detection is coupled to each of the four dNTPs via a linker to a terminal phosphate group. Using these modified nucleotides, sustained DNA synthesis by phi 29 DNA polymerase was observed, producing products up to several thousand bases in length, with the affinity of the labeled nucleotides and the DNA polymerization rate approaching those of the unmodified dNTPs. These results demonstrate that these nucleotides are suitable for single-molecule real-time DNA sequencing applications. [3]

C31H32N4O13S2

732.73

732.1407294

AF488 NHS ester;1374019-99-4

168679754

Orange to red solid powder

4

15

9

50

1610

0

CCN(CC)CC.C1CC(=O)N(C1=O)OC(=O)C2=CC(=C(C=C2)C3=C4C=CC(=[NH2+])C(=C4OC5=C3C=CC(=C5S(=O)(=O)[O-])N)S(=O)(=O)O)C(=O)O

OQTDJZAVCSDIGF-UHFFFAOYSA-N

InChI=1S/C25H17N3O13S2.C6H15N/c26-15-5-3-12-19(11-2-1-10(9-14(11)24(31)32)25(33)41-28-17(29)7-8-18(28)30)13-4-6-16(27)23(43(37,38)39)21(13)40-20(12)22(15)42(34,35)36;1-4-7(5-2)6-3/h1-6,9,26H,7-8,27H2,(H,31,32)(H,34,35,36)(H,37,38,39);4-6H2,1-3H3

3-amino-6-azaniumylidene-9-[2-carboxy-4-(2,5-dioxopyrrolidin-1-yl)oxycarbonylphenyl]-5-sulfoxanthene-4-sulfonate;N,N-diethylethanamine

AF488 NHS ester (TEA); AF488 NHS ester TEA; Triethylamine 2-(3,6-diamino-4-sulfo-5-sulfonatoxanthylium-9-yl)-5-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)benzoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O :~0.5 mg/mL (~0.68 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)