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.

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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).

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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.

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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.

Fluorescent probes enable detection of otherwise nonfluorescent species via highly sensitive laser-induced fluorescence. Organic amines are predominantly nonfluorescent and are of analytical interest in agricultural and food science, biomedical applications, and biowarfare detection. Alexa Fluor 488 N-hydroxysuccinimidyl ester (AF488 NHS-ester) is an amine-specific fluorescent probe. Here, we demonstrate low limit of detection of long-chain (C9 to C18) primary amines and optimize AF488 derivatization of long-chain primary amines. The reaction was found to be equally efficient in all solvents studied (dimethylsulfoxide, ethanol, and N,N-dimethylformamide). While an organic base (N,N-diisopropylethylamine) is required to achieve efficient reaction between AF488 NHS-ester and organic amines with longer hydrophobic chains, high concentrations (>5 mM) result in increased levels of ethylamine and propylamine in the blank. Optimal incubation times were found to be >12 hrs at room temperature. We present an initial capillary electrophoresis separation for analysis using a simple micellar electrokinetic chromatography (MEKC) buffer consisting of 12 mM sodium dodecylsulfate (SDS) and 5 mM carbonate, pH 10. Limits of detection using the optimized labeling conditions and these separation conditions were 5-17 nM. The method presented here represents a novel addition to the arsenal of fluorescent probes available for highly sensitive analysis of small organic molecules.[1]

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The growing integration of quality-by-design (QbD) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. Particularly valuable is the determination of the residual level of host cell proteins (HCPs) secreted, together with the product of interest, by the recombinant cells utilized for production. Though often referred to as a single impurity, HCPs comprise a variety of species with diverse abundance, size, function, and composition. The clearance of these impurities is a complex issue due to their cell line to cell line, product-to-product, and batch-to-batch variations. Improvements in HCP monitoring through proteomic-based methods have led to identification of a subset of "problematic" HCPs that are particularly challenging to remove, both at the product capture and product polishing steps, and compromise product stability and safety even at trace concentrations. This paper describes the development of synthetic peptide ligands capable of capturing a broad spectrum of Chinese hamster ovary (CHO) HCPs with a combination of peptide species that allow for advanced mixed-mode binding. Solid phase peptide libraries were screened for identification and characterization of peptides that capture CHO HCPs while showing minimal binding of human IgG, utilized here as a model product. Tetrameric and hexameric ligands featuring either multipolar or hydrophobic/positive amino acid compositions were found to be the most effective. Tetrameric multipolar ligands exhibited the highest targeted binding ratio (ratio of HCP clearance over IgG loss), more than double that of commercial mixed-mode and anion exchange resins utilized by industry for IgG polishing. All peptide resins tested showed preferential binding to HCPs compared to IgG, indicating potential uses in flow-through mode or weak-partitioning-mode chromatography.[2]

C25H17N3O13S2

631.54478430748

631.02

1374019-99-4

AF488 NHS ester TEA

154703996

Yellow to orange solid powder

-0.6

4

14

6

43

1590

0

C(C1C=C(C(=O)ON2C(CCC2=O)=O)C=CC=1C1=C2C=CC(N)=C(S(O)(=O)=O)C2=[O+]C2C(=C(N)C=CC1=2)S([O-])(=O)=O)(=O)O

FYCGGEVYPRTFDS-UHFFFAOYSA-N

InChI=1S/C25H17N3O13S2/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/h1-6,9,26H,7-8,27H2,(H,31,32)(H,34,35,36)(H,37,38,39)

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

AF488 NHS ester; 1374019-99-4; 3,6-Diamino-9-(2-carboxy-4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenyl)-5-sulfoxanthylium-4-sulfonate; HY-D1730; CS-0646332; G93909; 3-amino-6-azaniumylidene-9-[2-carboxy-4-(2,5-dioxopyrrolidin-1-yl)oxycarbonylphenyl]-5-sulfoxanthene-4-sulfonate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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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).

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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)

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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).

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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

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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.

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