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Alexa fluor 647 goat anti human igg

Manufactured by Thermo Fisher Scientific
Sourced in United States

Alexa Fluor 647 goat anti-human IgG is a fluorescently-labeled secondary antibody used for the detection of human immunoglobulin G (IgG) in various immunoassays and imaging applications. The antibody is conjugated with the Alexa Fluor 647 dye, which has excitation and emission maxima of 650 nm and 665 nm, respectively.

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31 protocols using alexa fluor 647 goat anti human igg

1

Immunogenicity of Pneumococcal Polysaccharide Conjugates

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Example 2

Binding experiments were performed by incubating microarray slides coated with the saccharides of general formula (I) with either a rabbit anti-SP8 typing serum or human pneumococcal reference serum 007sp (pooled sera of 287 humans immunized with Pneumovax® vaccine purchase from National Institute for Biological Standards and Control) in the dilutions indicated in the presence or absence of native SP8 polysaccharide, and using fluorescently labeled anti-rabbit (goat anti-rabbit IgG-FITC, abcam ab6717) or anti-human secondary antibodies (Alexa Fluor 488 goat anti-human IgM, Invitrogen A21215; Alexa Fluor 647 goat anti-human IgG, Invitrogen A21445).

Rabbits (n=3 per group) were immunized subcutaneously at multiple sites with glycoconjugates CRM197-18, CRM197-60 or CRM197-57 (10 μg glycan per dose) or CRM197 (100 μg) at days 0 and 14. Serum was collected at days 0, 14 and 21. The results of the immunization studies are summarized by FIG. 15.

All rabbits immunized with conjugates show a marked immune response against S. pneumoniae type 8 CPS-related oligosaccharides and S. pneumoniae type 8 CPS. Hence, all conjugates induce immunity against S. pneumoniae bacteria in rabbits.

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2

Profiling Cancer-Testis Antigens via ProtoArray

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ProtoArray microarrays (v4.0; Invitrogen) were purchased and used according to the manufacturer’s instructions. Briefly, after blocking for 1 h at 4° C and washing, arrays were incubated in Quadriperm dishes (Greiner Bio One) placed on a horizontal shaker (50 rpm) for 90 min at 4° C with individual sera diluted 1:500 in 5 ml washing buffer (0.1% Tween 20 [vol/vol], 1% BSA [wt/vol] in PBS). After washing, binding of IgG was detected by incubation with Alexa Fluor 647 goat anti-human IgG (Invitrogen) diluted 1:2,000 in assay buffer for 90 min at 4° C. Arrays were washed again and dried by centrifugation. Arrays were scanned at 10-μm resolution using a microarray scanner (Axon 4200AL with GenePix Pro Software; Molecular Devices), and fluorescence was detected according to the manufacturer’s instructions. Images were saved as 16-bit TIFF files and analysis was performed using GenePix. The median net intensity in relative fluorescence units (rfu) was reported for each spot. Out of 9,481 antigens, 77 cancer-testis antigens were selected and analyzed for pre- and post-vaccine expression in the sera (Table 4).
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3

Epigenetic Profiling of Pluripotent Stem Cells

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Primary antibodies used in this study include rabbit anti-5hmC (Active Motif, #39769), mouse anti-5mC (Active Motif, #39649), rabbit anti-NANOG (Abcam, ab80892), rabbit anti-H3K9me2 (Abcam, ab1220), rabbit anti-H3K9me3 (Abcam, ab8898), mouse anti-H3K27me3 (Wako, MABI0323), rabbit anti-H2AK119ub (Cell signaling, #8240), mouse anti-γH2AX (Sigma-Aldrich, 07–164), mouse anti-SCP3 (Santa Cruz, sc-74569), rabbit anti-SCP3 (Novus, NB-300-232), mouse anti-HP1γ (Invitrogen, MA3-054), human anti-Centromere Protein (CREST) (Antibodies Incorporated, 15–235), rabbit anti-TET1 (Millipore, 09–872), mouse anti-TET1 (Active Motif, 91172), rabbit anti-EZH2 (Cell signaling, #5246), rabbit anti-GFP antibody (Invitrogen, A11122), and rabbit anti-RING1B (Cell signaling, #5694). The secondary antibodies used in this study included Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 568 goat anti-mouse IgG, Alexa Fluor 568 goat anti-rabbit IgG, and Alexa Fluor 647 goat anti-human IgG (Invitrogen).
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4

Detection of Anti-Type I IFN Autoantibodies

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Detection of anti-type I IFN autoantibodies was performed using Gyros as described previously64 . Cytokines, recombinant human IFNα2 (Miltenyi Biotec, 130-108-984) or recombinant human IFNω (Merck, SRP3061) were first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, A39257), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody (Alexa Fluor 647 goat anti-human IgG (Thermo Fisher Scientific, A21445)) diluted in Rexxip F (Gyros Protein Technologies, P0004825; 1:500 dilution of the 2 mg ml−1 stock to yield a final concentration of 4 μg ml−1). Phosphate-buffered saline, 0.01% Tween-20 (PBS-T) and Gyros Wash buffer (Gyros Protein Technologies, P0020087) were prepared according to the manufacturer’s instructions. Plasma or serum samples were then diluted 1:100 in 0.01% PBS-T and tested with the Bioaffy 1000 CD (Gyros Protein Technologies, P0004253) and the Gyrolab xPand (Gyros Protein Technologies, P0020520). Cleaning cycles were performed in 20% ethanol.
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5

Quantifying Autoantibodies to Type I Interferons

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Recombinant E. coli-derived human (rh)GM-CSF (#215-GMP, R&D Systems) was first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (#A39257, Thermo Fisher Scientific), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody (Alexa Fluor 647 goat anti-human IgG (#A21445, Thermo Fisher Scientific)) diluted in Rexxip F (#P0004825, Gyros Protein Technologies; 1:500 dilution of the 2 mg/ml stock to yield a final concentration of 4 μg/ml). Buffer phosphate-buffered saline, 0.01% Tween 20 (PBS-T), and Gyros Wash buffer (#P0020087, Gyros Protein Technologies) were prepared according to the manufacturer’s instructions. Plasma or serum samples were then diluted 1:100 in 0.01% PBS-T and tested with the Bioaffy 1000 CD (#P0004253, Gyros Protein Technologies) and the Gyrolab xPand (#P0020520, Gyros Protein Technologies). Cleaning cycles were performed in 20% ethanol.
Screening of autoantibodies to type I IFN-α2a 100 pg/mL, IFN-β 10 ng/mL, and IFN-ω 100 pg/mL in plasma diluted 1 to 10 were performed as previously described using a dual interferon stimulated response element (ISRE) luciferase system in HEK293T cells [18 (link)]. Results were normalized to non-stimulated condition.
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6

Rapid Cytokine Quantification in Plasma

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Cytokines, recombinant human (rh)IFN-α2 (Milteny Biotec, ref. number 130-108-984) or rhIFN-ω (Merck, ref. number SRP3061), were first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, cat. number A39257), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody Alexa Fluor 647 goat anti-human IgG (Thermo Fisher Scientific, ref. number A21445) diluted in Rexip F (Gyros Protein Technologies, ref. number P0004825; 1/500 dilution of the 2 mg/mL stock to yield a final concentration of 4 μg/mL). Buffer PBS-T 0.01% and Gyros Wash buffer (Gyros Protein Technologies, ref. number P0020087) were prepared according to the manufacturer’s instructions. Plasma or serum samples were then diluted 1/100 in PBS-T 0.01% and tested with the Bioaffy 1000 CD (Gyros Protein Technologies, ref. number P0004253), and the Gyrolab X-Pand (Gyros Protein Technologies, ref. number P0020520). Cleaning cycles were performed in 20% ethanol.
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7

Biotinylated rhIFN-γ for Gyrolab Assay

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Recombinant human (rh) IFN-γ (R&D Systems, 285-IF-100/CF or 10067-IF-100) was first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, A39257), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody (Alexa Fluor 647 goat anti-human IgG [Thermo Fisher Scientific, A21445]) diluted in Rexxip F (Gyros Protein Technologies, P0004825; 1:500 dilution of the 2 mg/mL stock to yield a final concentration of 4 μg/mL). Buffer phosphate-buffered saline, 0.01% Tween 20 (PBS-T) and Gyros Wash buffer (Gyros Protein Technologies, P0020087) were prepared according to the manufacturer’s instructions. Plasma or serum samples were then diluted 1:100 in 0.01% PBS-T and tested with the Bioaffy 1000 CD (Gyros Protein Technologies, P0004253) and the Gyrolab xPand (Gyros Protein Technologies, P0020520). Cleaning cycles were performed in 20% ethanol. Threshold for positivity was determined by the average of the positive controls run on the day of the experiment in each run.
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8

Biotin-labeled rIFN detection assay

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Recombinant human (rh) IFNα2 (Miltenyi Biotec, reference number 130-108-984) or rhIFNω (Merck, reference number SRP3061), was first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, catalog number A39257), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody [Alexa Fluor 647 goat anti-human IgG (Thermo Fisher Scientific, reference number A21445)] diluted in Rexxip F (Gyros Protein Technologies, reference number P0004825; 1:500 dilution of the 2 mg/mL stock to yield a final concentration of 4 μg/mL). Buffer phosphate-buffered saline, 0.01% Tween 20 (PBS-T), and Gyros Wash buffer (Gyros Protein Technologies, reference number P0020087) were prepared according to the manufacturer’s instructions. Plasma or serum samples were then diluted 1:100 in PBS-T and tested with the Bioaffy 1000 CD (Gyros Protein Technologies, reference number P0004253) and the Gyrolab xPand (Gyros Protein Technologies, reference number P0020520).
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9

Detecting Cytokine Levels via Gyros Bioaffinity

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Cytokines, recombinant human (rh)IFN-α2 (Milteny Biotec, ref. number 130-108-984) or rhIFN-ω (Merck, ref. number SRP3061), were first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, cat. number A39257), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody (Alexa Fluor 647 goat anti-human IgG (Thermo Fisher Scientific, ref. number A21445) diluted in Rexip F (Gyros Protein Technologies, ref. number P0004825; 1/500 dilution of the 2 mg/mL stock to yield a final concentration of 4 μg/mL). Buffer PBS-T 0.01% and Gyros Wash buffer (Gyros Protein Technologies, ref. number P0020087) were prepared according to the manufacturer’s instructions. Plasma or serum samples were then diluted 1/100 in PBS-T 0.01% and tested with the Bioaffy 1000 CD (Gyros Protein Technologies, ref. number P0004253), and the Gyrolab X-Pand (Gyros Protein Technologies, ref. number P0020520). Cleaning cycles were performed in 20% ethanol.
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10

Bacterial Adhesion and Immunofluorescence Imaging

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Overnight bacterial cultures were diluted 1:200 in LB broth and 50 µl bacterial suspension was added per well in CellCarrier-96 Ultra plates (Perkin Elmer) and left in a static incubator at 37 °C for 2 h. The supernatant and any non-adhered bacteria were removed and the remaining bacteria were fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) for 15 min at room temperature, then washed with PBS. KM467 was diluted to 1 μg/ml in PBS + 1% bovine serum albumin (BSA) and added to the plate for 1 h (100 µl per well) at room temperature, after which it was replaced by 100 µl 2 μg/ml Alexa Fluor 647 Goat Anti-Human IgG (Thermo Fisher) plus 2 μg/ml DAPI (Thermo Fisher) in PBS + 1% BSA for 30 min in the dark. The plate was washed once with PBS, then 50 µl PBS was added to each well. The plates were imaged on an Opera Phenix using the Alexa Fluor 647 and DAPI channels and the 63 × water immersion objective and 16 fields and 3 Z-stacks were imaged per well.
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