Alexa fluor 647 labeling kit

Manufactured by Thermo Fisher Scientific

Sourced in Germany, United States

The Alexa Fluor 647 Labeling Kit is a product designed for the fluorescent labeling of proteins, antibodies, and other biomolecules. The kit provides the necessary reagents and protocols to enable efficient and stable conjugation of the Alexa Fluor 647 dye to target molecules. The core function of the kit is to facilitate the labeling process, allowing researchers to incorporate the fluorescent label into their samples for various analytical and imaging applications.

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Lab products found in correlation

Glutamax, by Thermo Fisher Scientific (4 mentions) Quantum alexa fluor 647 mesf molecules of equivalent soluble fluorochrome beads, by Bangs Laboratories (2 mentions) Quickcal program, by Bangs Laboratories (2 mentions) Macsquant vyb, by Miltenyi Biotec (2 mentions) Pen strep, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Annexin 5 pe 7 aad, by BD (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Mcl 1, by Cell Signaling Technology (1 mentions) Ez link micro sulfo nhs lc biotinylation kit, by Thermo Fisher Scientific (1 mentions) Normal rabbit serum, by Jackson ImmunoResearch (1 mentions) Hepatocyte culture medium, by Lonza (1 mentions) Liver perfusion medium, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by ScienCell (1 mentions) 96 well cell culture plate, by Corning (1 mentions) Clear immuno 384 well plate, by Thermo Fisher Scientific (1 mentions) Anti mouse cd146 antibody fitc, by Miltenyi Biotec (1 mentions) Anti mouse cd45 antibody vioblue, by Miltenyi Biotec (1 mentions) Alexa fluor 488 labeling kit, by Thermo Fisher Scientific (1 mentions) Pacific blue anti human cd31 antibody, by BioLegend (1 mentions)

17 protocols using alexa fluor 647 labeling kit

Labeling and Dilution of rITs

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HA22 (21 (link)) and LMB-11 (25 (link)) were produced as described.
rITs were labeled with Alexa Fluor 647 Labeling Kit (Invitrogen) according to manufacturer’s instructions. rITs for in vivo assays were diluted in phosphate buffered saline (PBS).
Secondary antibodies were purchased from Santa Cruz (Dallas, TX), primary antibodies (MCL1, PARP, EF2, GAPDH) from Cell Signaling (Danvers, MA), flow cytometry antibodies and Annexin V-PE/7-AAD from Becton Dickinson (Franklin Lakes, NJ).

Müller F., Cunningham T., Liu X.F., Wayne A.S, & Pastan I. (2016). Wide variability in the time required for immunotoxins to kill B lineage acute lymphoblastic leukemia cells: Implications for trial design. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(19), 4913-4922.

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Quantitative Serum Myl3 Assay for Rats

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Myl3 was quantitated in rat serum on a custom assay by MPI Research (Mattawan, MI). Samples (5μL) were analyzed in duplicate on Gyrolab™ Bioaffy 1000 CDs (Gyros, Uppsala, Sweden) using the Gyrolab xP workstation with a biotinylated (EZ Link™ Micro Sulfo-NHS-LC biotinylation kit, Thermo Scientific) capture antibody (BiosPacific, Emeryville, CA) and a fluorescently labeled (Alexa Fluor®647 labeling kit, Invitrogen) detection antibody (Abcam, Cambridge, UK). Prior to analysis, samples were diluted 1:4 in Rexxip A buffer (Gyros). Concentrations were interpolated from a 7-point standard curve using a recombinant; his-tagged rat specific myl3 expressed in-house (Eli Lilly and Company, Indianapolis, IN) as a calibrator and fit with a 5-parameter logistic curve. Capture antibody was diluted in PBS + 0.01% Tween 20, detection antibody was diluted in Rexxip F buffer (Gyros), and standards were diluted in Rexxip A. Prior to sample analysis, the performance of the assay was characterized using positive controls (assay calibrator spiked into rat serum) at six different concentrations (approximately mid-way between each standard concentration) ran on 3 different CDs on 3 different days to assess precision and accuracy. Myl3 was quantifiable from 0.05 ng/mL to 200 ng/mL with <12.5 %CV and −5 to −12% relative error.

Kim K., Chini N., Fairchild D.G., Engle S.K., Reagan W.J., Summers S.D, & Mirsalis J.C. (2016). Evaluation of Cardiac Toxicity Biomarkers in Rats from Different Laboratories. Toxicologic pathology, 44(8), 1072-1083.

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Quantifying EGFR and HER2 Receptor Density

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Receptor density studies were performed by flow cytometry on a MACSQuant VYB (Miltenyl Biotec) essentially as described20 (link). Briefly, anti-EGFR GA201 and anti-HER2 trastuzumab IgGs were first labeled with Alexa Fluor 647 labeling kit (Invitrogen) according to the supplier’s recommendations. Antibody concentration and fluorochrome to protein (F:P) ratio were determined by a ND-1000 spectrophotometer (NanoDrop). Parental NCI-H358 and NCI-H358.HER2.ko cells at ~4 × 10⁶ cells/mL were washed with ice-cold FACS Buffer (PBS pH 7.2, 2% FBS, 2 mM EDTA and 0.1% sodium azide) followed by incubation with the conjugated antibodies at saturating concentrations (≥ 20 μg/mL) for 30 min at 4 °C. After washing with FACS buffer, cells were fixed in ice-cold 1.8% paraformaldehyde (PFA) and detection of bound antibodies was performed on MACSQuant VYB using MACSQuantify™ software. Results were analyzed using the FlowJo analysis software (Tree Star). For quantitation of EGFR and HER2 receptor density on cells, Quantum Alexa Fluor 647 MESF (Molecules of Equivalent Soluble Fluorochrome) beads (Bangs Laboratories) were analyzed on the flow cytometer using similar settings to establish a standard curve. Using the QuickCal program (Bangs Laboratories) the calculated MESF was then divided by the antibody F:P ratio to give a corrected Antibody Binding Capacity (ABC).

Mazor Y., Sachsenmeier K.F., Yang C., Hansen A., Filderman J., Mulgrew K., Wu H, & Dall’Acqua W.F. (2017). Enhanced tumor-targeting selectivity by modulating bispecific antibody binding affinity and format valence. Scientific Reports, 7, 40098.

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Immunofluorescence and Proximity Ligation Assay Protocol

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HeLa cells were fixed with 100% methanol for EB1 immunostaining and PLA experiments (anti-EB1 antibody). All other immunostaining and PLA experiments were performed after cofixing cells with 4% paraformaldehyde, PHEM (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 4 mM MgCl₂, pH 6.9), and 0.5% Triton X-100 for 20 min. Immunofluorescence staining was performed in 3% BSA/TBS–0.05% Tween 20 using these antibodies anti-tubulin DM1-α, antiphospho-H3Ser10 (EMD Millipore), antiphospho–histone H3T3 (EMD Millipore), antiphospho-H2AT120 (Active Motif), phospho-KNL1 (S24 and S60; I.M. Cheeseman, Whitehead Institute for Biomedical Research, Cambridge, MA; Welburn et al., 2010 (link)), anti-AIM1 (Aurora B), anticentromere antigen (Antibodies, Inc.), anti-hEB1, and TO-PRO-3 (Invitrogen).
After the PLA procedure, cells were incubated in 10% normal mouse serum and 10% normal rabbit serum (Jackson ImmunoResearch Laboratories, Inc.) in 3% BSA/TBS + 0.05% Tween 20 for 30 min at RT to block any open binding sites on the PLA probes. FITC-conjugated anti–α-tubulin, DM1-α, and Alexa Fluor 647–conjugated polyclonal antibodies (xlNdc80, xlINCENP, or hBorealin) were used for costaining with PLA reactions. Alexa Fluor 647 polyclonal antibody conjugations were prepared using an Alexa Fluor 647 labeling kit following the manufacturer’s recommended protocol (Invitrogen).

Banerjee B., Kestner C.A, & Stukenberg P.T. (2014). EB1 enables spindle microtubules to regulate centromeric recruitment of Aurora B. The Journal of Cell Biology, 204(6), 947-963.

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Multispecies Immunophenotyping ELISA

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The following materials were purchased from commercial sources: Clear Immuno 384-Well Plate (Thermo Scientific, 8755), an anti-human IgG antibody (Bethyl Laboratories, A80-319A), Streptavidin-PolyHRP80 (Stereospecific Detection Technologies, SP80D50), Bovine Serum Albumin (BSA) for ELISA (Sigma-Aldrich, A7030), TMB Substrate (Surmodics, TMBS-1000–01), Carbonate-Bicarbonate Buffer capsule (Sigma-Aldrich, C3041), 96-well cell culture plate (Costar, 3799), BSA for cell assay (Sigma-Aldrich, A9418), Fetal bovine serum (FBS) (Sciencell, 0500), anti-mouse CD45 antibody-VioBlue (Miltenyi, 130-110-664), anti-mouse CD146 antibody-FITC (Miltenyi, 130-102-230), anti-human CD31 antibody-Pacific Blue (BioLegend, 303114), anti-non human primate CD45 antibody-APC (Miltenyi, 130-091-900), anti-CD32B (FcγRIIB) antibody (Sino Biological, 90014-R046), Liver Perfusion Medium (Gibco, 17701-038), Endothelial cell medium (Sciencell, 1001), Hepatocyte Culture Medium (Lonza, CC-3198), Alexa Fluor 488 Labeling Kit (Invitrogen, A20181), Alexa Fluor 647 Labeling Kit (Invitrogen, A20173). Other reagents were purchased from local commercial sources.

Noguchi Y., Ozeki K., Takesue H, & Akita H. (2021). A cell based assay for evaluating binding and uptake of an antibody using hepatic nonparenchymal cells. Scientific Reports, 11, 8383.

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Flow Cytometric Analysis of Antibody Binding

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RITs were labeled with the Alexa Fluor 647 Labeling Kit (Invitrogen) for 3.5 hrs and purified according to manufacturer’s instructions. Harvested cells were incubated for 30, 60 and 120 min at 37°C with 2 μg/ml of SS1P-Alexa647 or RG7787-Alexa647 and processed as previously described (17 (link)). Fluorescence intensity was analyzed on a FACSCalibur.

Hollevoet K., Mason-Osann E., Liu X.F., Imhof-Jung S., Niederfellner G, & Pastan I. (2014). In vitro and in vivo activity of the low-immunogenic anti-mesothelin immunotoxin RG7787 in pancreatic cancer. Molecular cancer therapeutics, 13(8), 2040-2049.

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Monoclonal Antibody Purification and Labeling

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Hybridoma cell lines were adapted to CD Hybridoma AGT medium (Gibco); a serum-free media (SFM), supplemented with 40 mL/L GlutaMAX (ThermoFisher). Supernatants were harvested; mAbs were purified on an rProtein A GraviTrap column (GE Healthcare). mAbs were concentrated and buffer-exchanged (Millipore) to PBS. Glycerol was added to 50% and the purified mAbs were stored at -20°C. Each mAb was quantified by densitometry using ImageJ (27 (link)) and/or by a BCA protein assay. mAb light and heavy chains were verified using a non-reducing SDS-polyacrylamide gel (Bio-Rad). mAbs were either used unlabeled or fluorescently-labelled with an Alexa Fluor 647 labeling kit (Invitrogen).
The anti-CD30 mAb clone AC10, which is the mAb component of brentuximab, was obtained from AdipoGen (unlabeled or APC-labeled). Anti-CD30 clone BerH2 (PE-labelled) was acquired from Invitrogen. Anti-CD20-PE and anti-CXCR3-Alexa Fluor 700 were purchased from BioLegend as were all isotype control antibodies. Goat anti-mouse IgG (H+L) secondary antibody labeled with Alexa Fluor 647 was purchased from ThermoFisher.

Faber M.L., Oldham R.A., Thakur A., Rademacher M.J., Kubicka E., Dlugi T.A., Gifford S.A., McKillop W.M., Schloemer N.J., Lum L.G, & Medin J.A. (2023). Novel anti-CD30/CD3 bispecific antibodies activate human T cells and mediate potent anti-tumor activity. Frontiers in Immunology, 14, 1225610.

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Quantifying Cell Surface Receptor Density

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Receptor density studies were performed by flow cytometry on a MACSQuant VYB (Miltenyl Biotec) essentially as described [27 (link)]. Briefly, anti-CD4 (ibalizumab), anti-EGFR (GA201) and anti-HER2 (B1D2) IgGs were first labeled with Alexa Fluor 647 labeling kit (Invitrogen) according to the manufacturer’s instructions. Antibody concentration and fluorochrome to protein (F:P) ratio were calculated by a ND-1000 spectrophotomer (NanoDrop). Cells at ~ 4 × 10⁶ cells/mL were first washed with ice-cold FACS Buffer (PBS pH 7.2, 2% FBS, 2 mM EDTA and 0.1% sodium azide) followed by incubation with saturating concentration (≥ 20 μg/mL) of conjugated antibodies for 30 min at 4°C. After washing with FACS buffer, cells were fixed in ice-cold 1.8% paraformaldehyde (PFA) and detection of bound antibodies was determined on MACSQuant VYB using MACSQuantify™ software. Data were analyzed with the FlowJo analysis software. For quantitation of CD4, EGFR and HER2 density on cells, Quantum Alexa Fluor 647 MESF (Molecules of Equivalent Soluble Fluorochrome) beads (Bangs Laboratories) were processed on the flow cytometer using similar settings. QuickCal program (Bangs Laboratories) was used to establish a standard curve. The calculated MESF was then divided by the antibody F:P ratio to give a corrected Antibody Binding Capacity (ABC).

Mazor Y., Yang C., Borrok M.J., Ayriss J., Aherne K., Wu H, & Dall’Acqua W.F. (2016). Enhancement of Immune Effector Functions by Modulating IgG’s Intrinsic Affinity for Target Antigen. PLoS ONE, 11(6), e0157788.

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Quantifying Mesothelin Expression and RIT Internalization

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To quantify the cell surface mesothelin expression, cells were grown for 2 days, harvested, washed with FACS buffer (PBS with 5% FBS and 0.1% NaN₃), and incubated with 5 μg/mL of mouse antimesothelin MN antibody^{23 (link)} (Rockland Immunochemicals Inc., Gilbertsville, PA) on ice for 30 minutes. After washing, cells were incubated with goat antimouse IgG-R-PE (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) on ice for 30 minutes in the dark, and washed again twice. Mesothelin expression was analyzed on a FACSCalibur. QuantiBRITE PE beads (BD Pharmingen) were used to quantitate the number of mesothelin sites per cell. To evaluate RIT internalization and the effect of ABT-737 on uptake, cells were incubated for 3 hours at 37°C with 2 μg/mL of SS1P-Alexa647 alone or in combination with 10 μM ABT-737. SS1P was labeled with the Alexa Fluor 647 Labeling Kit (Invitrogen) according to manufacturer’s instructions. After incubation, cells were washed with FACS buffer, and surface-bound SS1P was removed by stripping cells with glycine buffer (0.2 mol/L glycine, pH 2.5 and 1 mg/mL of BSA) for 10 minutes, neutralized with Tris-HCl 1M pH 8, and washed with FACS buffer. Fluorescence intensity was analyzed on a FACSCalibur.

Hollevoet K., Antignani A., FitzGerald D.J, & Pastan I. (2014). Combining the Antimesothelin Immunotoxin SS1P With the BH3-mimetic ABT-737 Induces Cell Death in SS1P-resistant Pancreatic Cancer Cells. Journal of immunotherapy (Hagerstown, Md. : 1997), 37(1), 8-15.

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Quantifying Mesothelin-Targeted Internalization

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RITs were labeled with the Alexa Fluor 647 Labeling Kit (Invitrogen) for 3.5 hrs and purified according to manufacturer’s instructions. Harvested cells were incubated for 30, 75 and 150 min at 37°C with a saturating 2 μg/ml of SS1P-Alexa647 or RG7787-Alexa647 and processed as previously described [22 (link)]. Fluorescence intensity was analyzed on a FACSCalibur. Uptake was expressed in number of internalized RG7787 molecules, which was calculated by assuming the RG7787-Alexa647 geomean surface expression of KLM-1 equal to 60 x 10³ RG7787 molecules (= surface mesothelin binding sites per KLM-1 cell, as evaluated by flow cytometry and QuantiBRITE R-PE beads).

Hollevoet K., Mason-Osann E., Müller F, & Pastan I. (2015). Methylation-Associated Partial Down-Regulation of Mesothelin Causes Resistance to Anti-Mesothelin Immunotoxins in a Pancreatic Cancer Cell Line. PLoS ONE, 10(3), e0122462.

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