goat anti rabbit igg alexa 488 secondary antibody  (Thermo Fisher)


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    Name:
    Goat anti Rabbit IgG H L Secondary Antibody
    Description:
    Goat anti Rabbit IgG H L Secondary Antibody for Western Blot IF ICC IHC Flow IP
    Catalog Number:
    31210
    Price:
    None
    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher goat anti rabbit igg alexa 488 secondary antibody
    Confirmation of the formation of squid protein-based architectures in human cells. a  (Left) Merged fluorescence microscopy images of fixed RfA1-expressing cells stained with DAPI and labeled with an antibody pair specific for the protein’s histidine-tag. (Right) Merged fluorescence microscopy images of fixed RfA1-expressing cells stained with DAPI and labeled with an antibody pair specific for reflectins’ unique sequence. The signals corresponding to DAPI and the Alexa 488 fluorophore-conjugated secondary antibody are colored blue and green, respectively, in both images. The scale bars are 15 µm in both images.  b  (Top) A TEM image of a cross-section from one representative RfA1-expressing cell, which reveals the presence of electron-dense structures. The scale bar is 2 µm. The inset shows a close-up image of a nanoparticle cluster. The scale bar is 500 nm for the inset. (Bottom) A TEM image of a cross-section from another representative RfA1-expressing cell, which also reveals the presence of electron-dense structures. The scale bar is 2 µm. The inset shows a close-up image of a nanoparticle cluster. The scale bar is 500 nm for the inset.  c  Merged fluorescence microscopy images of fixed RfA1- and RFP-expressing cells stained with DAPI and labeled with an antibody pair specific for reflectins’ unique sequence. (Left) The signals corresponding to DAPI and the Alexa 488 fluorophore-conjugated secondary antibody are shown, and they are colored blue and green, respectively. (Right) The signals corresponding to DAPI, the Alexa 488 fluorophore-conjugated secondary antibody, and RFP are shown, and they are colored blue, green, and red, respectively. The scale bars are 10 µm in both images.  d  (Left) An immuno-EM image of a cross-section from a representative cell, which has been labeled with a primary antibody specific for reflectins’ unique sequence followed by a complementary secondary antibody conjugated to a gold nanoparticle. The scale bar is 2 µm. (Right) A close-up image of a small cluster of RfA1 structures (large gray spheres), which are specifically labeled by antibody-conjugated gold nanoparticles (small black dots). The scale bar is 500 nm for the inset.
    Goat anti Rabbit IgG H L Secondary Antibody for Western Blot IF ICC IHC Flow IP
    https://www.bioz.com/result/goat anti rabbit igg alexa 488 secondary antibody/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti rabbit igg alexa 488 secondary antibody - by Bioz Stars, 2021-03
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    Images

    1) Product Images from "Cephalopod-inspired optical engineering of human cells"

    Article Title: Cephalopod-inspired optical engineering of human cells

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16151-6

    Confirmation of the formation of squid protein-based architectures in human cells. a  (Left) Merged fluorescence microscopy images of fixed RfA1-expressing cells stained with DAPI and labeled with an antibody pair specific for the protein’s histidine-tag. (Right) Merged fluorescence microscopy images of fixed RfA1-expressing cells stained with DAPI and labeled with an antibody pair specific for reflectins’ unique sequence. The signals corresponding to DAPI and the Alexa 488 fluorophore-conjugated secondary antibody are colored blue and green, respectively, in both images. The scale bars are 15 µm in both images.  b  (Top) A TEM image of a cross-section from one representative RfA1-expressing cell, which reveals the presence of electron-dense structures. The scale bar is 2 µm. The inset shows a close-up image of a nanoparticle cluster. The scale bar is 500 nm for the inset. (Bottom) A TEM image of a cross-section from another representative RfA1-expressing cell, which also reveals the presence of electron-dense structures. The scale bar is 2 µm. The inset shows a close-up image of a nanoparticle cluster. The scale bar is 500 nm for the inset.  c  Merged fluorescence microscopy images of fixed RfA1- and RFP-expressing cells stained with DAPI and labeled with an antibody pair specific for reflectins’ unique sequence. (Left) The signals corresponding to DAPI and the Alexa 488 fluorophore-conjugated secondary antibody are shown, and they are colored blue and green, respectively. (Right) The signals corresponding to DAPI, the Alexa 488 fluorophore-conjugated secondary antibody, and RFP are shown, and they are colored blue, green, and red, respectively. The scale bars are 10 µm in both images.  d  (Left) An immuno-EM image of a cross-section from a representative cell, which has been labeled with a primary antibody specific for reflectins’ unique sequence followed by a complementary secondary antibody conjugated to a gold nanoparticle. The scale bar is 2 µm. (Right) A close-up image of a small cluster of RfA1 structures (large gray spheres), which are specifically labeled by antibody-conjugated gold nanoparticles (small black dots). The scale bar is 500 nm for the inset.
    Figure Legend Snippet: Confirmation of the formation of squid protein-based architectures in human cells. a (Left) Merged fluorescence microscopy images of fixed RfA1-expressing cells stained with DAPI and labeled with an antibody pair specific for the protein’s histidine-tag. (Right) Merged fluorescence microscopy images of fixed RfA1-expressing cells stained with DAPI and labeled with an antibody pair specific for reflectins’ unique sequence. The signals corresponding to DAPI and the Alexa 488 fluorophore-conjugated secondary antibody are colored blue and green, respectively, in both images. The scale bars are 15 µm in both images. b (Top) A TEM image of a cross-section from one representative RfA1-expressing cell, which reveals the presence of electron-dense structures. The scale bar is 2 µm. The inset shows a close-up image of a nanoparticle cluster. The scale bar is 500 nm for the inset. (Bottom) A TEM image of a cross-section from another representative RfA1-expressing cell, which also reveals the presence of electron-dense structures. The scale bar is 2 µm. The inset shows a close-up image of a nanoparticle cluster. The scale bar is 500 nm for the inset. c Merged fluorescence microscopy images of fixed RfA1- and RFP-expressing cells stained with DAPI and labeled with an antibody pair specific for reflectins’ unique sequence. (Left) The signals corresponding to DAPI and the Alexa 488 fluorophore-conjugated secondary antibody are shown, and they are colored blue and green, respectively. (Right) The signals corresponding to DAPI, the Alexa 488 fluorophore-conjugated secondary antibody, and RFP are shown, and they are colored blue, green, and red, respectively. The scale bars are 10 µm in both images. d (Left) An immuno-EM image of a cross-section from a representative cell, which has been labeled with a primary antibody specific for reflectins’ unique sequence followed by a complementary secondary antibody conjugated to a gold nanoparticle. The scale bar is 2 µm. (Right) A close-up image of a small cluster of RfA1 structures (large gray spheres), which are specifically labeled by antibody-conjugated gold nanoparticles (small black dots). The scale bar is 500 nm for the inset.

    Techniques Used: Fluorescence, Microscopy, Expressing, Staining, Labeling, Sequencing, Transmission Electron Microscopy

    Related Articles

    Immunofluorescence:

    Article Title: Enhanced connexin 43 expression delays intra-mitoitc duration and cell cycle traverse independently of gap junction channel function
    Article Snippet: Western blot primary antibodies were visualized using anti mouse or rabbit secondary antibodies as required: for chemiluminescent detection horse radish peroxidise (HRP) (BioRad) was used and for detection using the LiCor Odyssey imaging system IRDye-680/800 (LiCor) secondary antibodies were used. .. For immunofluorescence primary antibodies were visualised using goat anti mouse-Alexa 488 or goat anti rabbit-Alexa 594 secondary antibodies as required (Invitrogen). ..

    Blocking Assay:

    Article Title: The Yeast Split-Ubiquitin Membrane Protein Two-Hybrid Screen Identifies BAP31 as a Regulator of the Turnover of Endoplasmic Reticulum-Associated Protein Tyrosine Phosphatase-Like B
    Article Snippet: The cells were briefly permeabilized in phosphate-buffered saline-10% fetal calf serum-0.2% Triton X-100 and then treated with blocking solution (phosphate-buffered saline containing 10% fetal calf serum and 0.1% Triton X-100). .. Antibody incubations were conducted in blocking solution for 1 h at room temperature with the indicated primary antibodies and either goat anti-mouse immunoglobulin G or goat anti-rabbit immunoglobulin G secondary antibodies coupled to Alexa 488 (green) or Alexa 594 (red) (Molecular Probes). .. Cells were visualized by confocal or conventional fluorescence microscopy.

    other:

    Article Title: In vitro and in vivo inhibition of breast cancer cell growth by targeting the Hedgehog/GLI pathway with SMO (GDC-0449) or GLI (GANT-61) inhibitors
    Article Snippet: Goat anti-rabbit IgG Alexa fluor-594-conjugated and goat anti-mouse IgG Alexa fluor-488-conjugated secondary antibody came from Invitrogen (Milano, Italy).

    Incubation:

    Article Title: Function of Lipid Storage Droplet 1 (Lsd1) in Wing Development of Drosophila melanogaster
    Article Snippet: Samples were then incubated with rabbit anti-cleaved caspase-3 IgG (Cell Signaling Technology, Tokyo, Japan) at a 1:100 dilution for 16 h at 4 °C. .. After extensive washing with PBST, the samples were incubated with goat anti-rabbit IgG Alexa Fluor™ 488 (Molecular Probes, Invitrogen) at a 1:400 dilution for 2 h at 25 °C, washed with PBST and PBS, and then mounted in Vectashield mounting medium. .. Preparations were examined under a fluorescence BX-50 microscope equipped with a cooled CCD camera.

    Article Title: Characterization of novel microneme adhesive repeats (MAR) in Eimeria tenella
    Article Snippet: Immunofluorescence MDBK monolayers infected for 4 h with E. tenella sporozoites were fixed in 4% paraformaldehyde in PBS for 15 min and washed in PBS. .. Fixed monolayers were blocked by incubating in 3% BSA, 0.25% Triton ×100 in PBS for 30 min then in rabbit anti-EtMIC2 serum (1/500) for 1 h. After three washes in PBS, monolayers were incubated with goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1/1000; Invitrogen), washed in PBS, then observed under UV fluorescence using a Leica DMI3000B microscope and photographed with a Leica DCF365FX camera. ..

    Fluorescence:

    Article Title: Characterization of novel microneme adhesive repeats (MAR) in Eimeria tenella
    Article Snippet: Immunofluorescence MDBK monolayers infected for 4 h with E. tenella sporozoites were fixed in 4% paraformaldehyde in PBS for 15 min and washed in PBS. .. Fixed monolayers were blocked by incubating in 3% BSA, 0.25% Triton ×100 in PBS for 30 min then in rabbit anti-EtMIC2 serum (1/500) for 1 h. After three washes in PBS, monolayers were incubated with goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1/1000; Invitrogen), washed in PBS, then observed under UV fluorescence using a Leica DMI3000B microscope and photographed with a Leica DCF365FX camera. ..

    Microscopy:

    Article Title: Characterization of novel microneme adhesive repeats (MAR) in Eimeria tenella
    Article Snippet: Immunofluorescence MDBK monolayers infected for 4 h with E. tenella sporozoites were fixed in 4% paraformaldehyde in PBS for 15 min and washed in PBS. .. Fixed monolayers were blocked by incubating in 3% BSA, 0.25% Triton ×100 in PBS for 30 min then in rabbit anti-EtMIC2 serum (1/500) for 1 h. After three washes in PBS, monolayers were incubated with goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1/1000; Invitrogen), washed in PBS, then observed under UV fluorescence using a Leica DMI3000B microscope and photographed with a Leica DCF365FX camera. ..

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    Thermo Fisher rabbit anti goat alexa 488 antibody
    Establishment of the optimal printing and storage conditions for biotinylated antigen arrays in streptavidin-coated 96-well plates. (A) Schematic representation of a 96 well plate with a 5 × 5 antigen array. (B–L) A purified biotinylated antibody (OX68-bio) was spotted in streptavidin-coated 96 well plates. (B and C) Detection with either a non-fluorescent alkaline phosphatase-conjugated secondary antibody followed by a precipitating colourimetric NBT/BCIP substrate (B); and, with a fluorescent Alexa 568-conjugated secondary antibody (C). (D) Different concentrations of OX68-bio, 1 = 0.5; 2 = 0.25; 3 = 0.1; 4 = 0.05 μg/μl were arranged vertically, and in quadruplicate horizontally. (E) Different amounts of Tween 20 added prior to printing, 1 = 10%; 2 = 1%; 3 = 0.1%; 4 = None. (F) Different amounts of glycerol added prior to printing, 1 = 10%; 2 = 1%; 3 = 0.1%; 4 = 1% Tween 20. (G) Incubation of different concentrations of spotted antibody (as in (D)) for 2 h at room temperature with a 4% formalin solution. Storage of a printed plate for 2 days at room temperature (H), and at −20 °C (I), prior to antigen detection. Examples of: a 25-spot array using 0.4 mm pins, 0.66 mm pitch (J); a 53-spot array using 0.2 mm pins, 0.45 mm pitch (K), and a 101-spot array using 0.2 mm pins, 0.32 mm pitch. In panels D–L printed antibody was detected with an <t>Alexa-488</t> conjugated secondary antibody.
    Rabbit Anti Goat Alexa 488 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti goat alexa 488 antibody/product/Thermo Fisher
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti goat alexa 488 antibody - by Bioz Stars, 2021-03
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    97
    Thermo Fisher rabbit antibody alexa 488
    Competitive binding of rDIII and EGF.  (A) Flow cytometry. MDA-MB-231 cells were incubated with 2.00 μM rDIII, in the presence of increasing concentrations of EGF (0.45, 0.85, 1.25, and 2.00 μM). rDIII binding to the cell surface was detected with anti-His tag and Alexa ®  488 antibodies. The fluorescence signal for rDIII gradually decreases with increasing EGF concentrations. (B) Displacement of cell surface-bound I 125 -EGF by rDIII. MDA-MB-231 cells were incubated with I0.5 nM  125 -EGF and increasing concentrations of cold rDIII (top) or EGF (bottom). The 0.5 nM (≈0.15 μCi) working concentration of I 125 -EGF was determined by calculating the specific binding of I 125 EGF (“specific”) based on total and nonspecific binding of I 125 -EGF to MDA-MB-231 cells (inset in bottom panel). Cells were incubated with increasing concentrations of I 125 -EGF in the absence (total binding; “total”) or presence of an excess amount (330 nM) of unlabeled EGF (nonspecific binding; “nonspecific”).
    Rabbit Antibody Alexa 488, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit antibody alexa 488/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit antibody alexa 488 - by Bioz Stars, 2021-03
    97/100 stars
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    Image Search Results


    Establishment of the optimal printing and storage conditions for biotinylated antigen arrays in streptavidin-coated 96-well plates. (A) Schematic representation of a 96 well plate with a 5 × 5 antigen array. (B–L) A purified biotinylated antibody (OX68-bio) was spotted in streptavidin-coated 96 well plates. (B and C) Detection with either a non-fluorescent alkaline phosphatase-conjugated secondary antibody followed by a precipitating colourimetric NBT/BCIP substrate (B); and, with a fluorescent Alexa 568-conjugated secondary antibody (C). (D) Different concentrations of OX68-bio, 1 = 0.5; 2 = 0.25; 3 = 0.1; 4 = 0.05 μg/μl were arranged vertically, and in quadruplicate horizontally. (E) Different amounts of Tween 20 added prior to printing, 1 = 10%; 2 = 1%; 3 = 0.1%; 4 = None. (F) Different amounts of glycerol added prior to printing, 1 = 10%; 2 = 1%; 3 = 0.1%; 4 = 1% Tween 20. (G) Incubation of different concentrations of spotted antibody (as in (D)) for 2 h at room temperature with a 4% formalin solution. Storage of a printed plate for 2 days at room temperature (H), and at −20 °C (I), prior to antigen detection. Examples of: a 25-spot array using 0.4 mm pins, 0.66 mm pitch (J); a 53-spot array using 0.2 mm pins, 0.45 mm pitch (K), and a 101-spot array using 0.2 mm pins, 0.32 mm pitch. In panels D–L printed antibody was detected with an Alexa-488 conjugated secondary antibody.

    Journal: Biochemical and Biophysical Research Communications

    Article Title: Development of an antigen microarray for high throughput monoclonal antibody selection

    doi: 10.1016/j.bbrc.2013.12.033

    Figure Lengend Snippet: Establishment of the optimal printing and storage conditions for biotinylated antigen arrays in streptavidin-coated 96-well plates. (A) Schematic representation of a 96 well plate with a 5 × 5 antigen array. (B–L) A purified biotinylated antibody (OX68-bio) was spotted in streptavidin-coated 96 well plates. (B and C) Detection with either a non-fluorescent alkaline phosphatase-conjugated secondary antibody followed by a precipitating colourimetric NBT/BCIP substrate (B); and, with a fluorescent Alexa 568-conjugated secondary antibody (C). (D) Different concentrations of OX68-bio, 1 = 0.5; 2 = 0.25; 3 = 0.1; 4 = 0.05 μg/μl were arranged vertically, and in quadruplicate horizontally. (E) Different amounts of Tween 20 added prior to printing, 1 = 10%; 2 = 1%; 3 = 0.1%; 4 = None. (F) Different amounts of glycerol added prior to printing, 1 = 10%; 2 = 1%; 3 = 0.1%; 4 = 1% Tween 20. (G) Incubation of different concentrations of spotted antibody (as in (D)) for 2 h at room temperature with a 4% formalin solution. Storage of a printed plate for 2 days at room temperature (H), and at −20 °C (I), prior to antigen detection. Examples of: a 25-spot array using 0.4 mm pins, 0.66 mm pitch (J); a 53-spot array using 0.2 mm pins, 0.45 mm pitch (K), and a 101-spot array using 0.2 mm pins, 0.32 mm pitch. In panels D–L printed antibody was detected with an Alexa-488 conjugated secondary antibody.

    Article Snippet: After washing with PBT (PBS + 0.1% Tween), arrays were incubated for 2 hours with a goat anti-mouse Alexa 488 secondary antibody (Invitrogen), washed in PBT, followed by a rabbit anti-goat Alexa 488 antibody (Invitrogen).

    Techniques: Purification, Incubation

    BoHV-1 infection increases the number and length of TNT connections. KOP cells and bovine fibroblasts were grown for 1 day on gelatin-coated coverslips and infected with BoHV-1-WT at an MOI of 1. Control cells were left uninfected. At 18 h.p.i., cells were fixed and stained with Alexa Fluor 488-phalloidin conjugate to visualize the actin cytoskeleton. The number and length of cell connections were quantified using confocal microscopy. TNT connections between infected and uninfected cells were manually counted and measured in 6 separate experiments. Data were collected using the Lucia program. (A) Comparison of the frequency of nanotubes formation in BoHV-1-infected and in uninfected cells. (B) Comparison of TNT length. (C) Analysis of ratio of short (≤20 μm) to long ( > 20 μm) TNTs showed a significant shift toward long connections upon BoHV-1 infection. Data collected in 6 separate experiments represent the means, normalized to the value for control (uninfected) cells, arbitrarily set at 100%. *, P

    Journal: Journal of Virology

    Article Title: Tunneling Nanotubes as a Novel Route of Cell-to-Cell Spread of Herpesviruses

    doi: 10.1128/JVI.00090-18

    Figure Lengend Snippet: BoHV-1 infection increases the number and length of TNT connections. KOP cells and bovine fibroblasts were grown for 1 day on gelatin-coated coverslips and infected with BoHV-1-WT at an MOI of 1. Control cells were left uninfected. At 18 h.p.i., cells were fixed and stained with Alexa Fluor 488-phalloidin conjugate to visualize the actin cytoskeleton. The number and length of cell connections were quantified using confocal microscopy. TNT connections between infected and uninfected cells were manually counted and measured in 6 separate experiments. Data were collected using the Lucia program. (A) Comparison of the frequency of nanotubes formation in BoHV-1-infected and in uninfected cells. (B) Comparison of TNT length. (C) Analysis of ratio of short (≤20 μm) to long ( > 20 μm) TNTs showed a significant shift toward long connections upon BoHV-1 infection. Data collected in 6 separate experiments represent the means, normalized to the value for control (uninfected) cells, arbitrarily set at 100%. *, P

    Article Snippet: Secondary goat anti-rabbit IgG Alexa Fluor 488 and rabbit anti-goat IgG Alexa Fluor 488 conjugates (Molecular Probes) were used at a 1:2,000 dilution.

    Techniques: Infection, Staining, Confocal Microscopy

    SH2-Bβ shuttles between the cytoplasm and nucleus. (A) PC12 cells were transiently transfected with cDNA encoding GFP-SH2-Bβ or GFP. Eighteen hours after transfection, cells were either mock treated (left panels) or treated with 20 nM leptomycin B (+LMB) for 3 h (right panels) before fixation. Images were then taken to determine the subcellular distribution of GFP-SH2-Bβ. DAPI images next to the fluorescence images showed the localization of the nucleus. (B) COS-7 cells were transiently transfected with GFP-SH2-Bβ or GFP. Eighteen hours after transfection, cells were either mock treated (left panels) or treated with 20 nM LMB for 5 h (right panels) before fixation. Images were then taken using epifluorescence microscopy. DAPI images showed the nucleus localization. (C) Quantification of GFP-SH2-Bβ distribution in the cytoplasm (C) or in the cytoplasm and nucleus (C+N) in either PC12 cells or COS-7 cells without or with LMB (+ LMB) treatment. A total of 17 to 43 cells were counted from two experiments per condition. (D) PC12 cells were incubated without or with LMB for 3h before fixation. Endogenous SH2-Bβ was detected by incubating cells with goat anti-SH2-Bβ antibody and then with rabbit anti-goat Alexa 488. The images were visualized using epifluorescence microscopy. The corresponding DAPI images show the nuclei. (E) COS-7 cells were incubated without or with LMB for 28 h before fixation. Endogenous SH2-Bβ was detected and visualized as described for panel D.

    Journal: Molecular and Cellular Biology

    Article Title: Adapter Protein SH2-B? Undergoes Nucleocytoplasmic Shuttling: Implications for Nerve Growth Factor Induction of Neuronal Differentiation

    doi: 10.1128/MCB.24.9.3633-3647.2004

    Figure Lengend Snippet: SH2-Bβ shuttles between the cytoplasm and nucleus. (A) PC12 cells were transiently transfected with cDNA encoding GFP-SH2-Bβ or GFP. Eighteen hours after transfection, cells were either mock treated (left panels) or treated with 20 nM leptomycin B (+LMB) for 3 h (right panels) before fixation. Images were then taken to determine the subcellular distribution of GFP-SH2-Bβ. DAPI images next to the fluorescence images showed the localization of the nucleus. (B) COS-7 cells were transiently transfected with GFP-SH2-Bβ or GFP. Eighteen hours after transfection, cells were either mock treated (left panels) or treated with 20 nM LMB for 5 h (right panels) before fixation. Images were then taken using epifluorescence microscopy. DAPI images showed the nucleus localization. (C) Quantification of GFP-SH2-Bβ distribution in the cytoplasm (C) or in the cytoplasm and nucleus (C+N) in either PC12 cells or COS-7 cells without or with LMB (+ LMB) treatment. A total of 17 to 43 cells were counted from two experiments per condition. (D) PC12 cells were incubated without or with LMB for 3h before fixation. Endogenous SH2-Bβ was detected by incubating cells with goat anti-SH2-Bβ antibody and then with rabbit anti-goat Alexa 488. The images were visualized using epifluorescence microscopy. The corresponding DAPI images show the nuclei. (E) COS-7 cells were incubated without or with LMB for 28 h before fixation. Endogenous SH2-Bβ was detected and visualized as described for panel D.

    Article Snippet: Fixed cells were incubated with anti-SH2-Bβ antibody (Santa Cruz) (diluted 1:25) for 1 h, with rabbit anti-goat Alexa 488 antibody (Fab′) (Molecular Probe) (diluted 1:100) for 1 h, and then with 4′,6′-diamidino-2-phenylindole (DAPI) (Molecular Probe) (2 ng/ml) for 5 min.

    Techniques: Transfection, Fluorescence, Epifluorescence Microscopy, Incubation

    Competitive binding of rDIII and EGF.  (A) Flow cytometry. MDA-MB-231 cells were incubated with 2.00 μM rDIII, in the presence of increasing concentrations of EGF (0.45, 0.85, 1.25, and 2.00 μM). rDIII binding to the cell surface was detected with anti-His tag and Alexa ®  488 antibodies. The fluorescence signal for rDIII gradually decreases with increasing EGF concentrations. (B) Displacement of cell surface-bound I 125 -EGF by rDIII. MDA-MB-231 cells were incubated with I0.5 nM  125 -EGF and increasing concentrations of cold rDIII (top) or EGF (bottom). The 0.5 nM (≈0.15 μCi) working concentration of I 125 -EGF was determined by calculating the specific binding of I 125 EGF (“specific”) based on total and nonspecific binding of I 125 -EGF to MDA-MB-231 cells (inset in bottom panel). Cells were incubated with increasing concentrations of I 125 -EGF in the absence (total binding; “total”) or presence of an excess amount (330 nM) of unlabeled EGF (nonspecific binding; “nonspecific”).

    Journal: The Journal of Cell Biology

    Article Title: Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution

    doi: 10.1083/jcb.200208145

    Figure Lengend Snippet: Competitive binding of rDIII and EGF. (A) Flow cytometry. MDA-MB-231 cells were incubated with 2.00 μM rDIII, in the presence of increasing concentrations of EGF (0.45, 0.85, 1.25, and 2.00 μM). rDIII binding to the cell surface was detected with anti-His tag and Alexa ® 488 antibodies. The fluorescence signal for rDIII gradually decreases with increasing EGF concentrations. (B) Displacement of cell surface-bound I 125 -EGF by rDIII. MDA-MB-231 cells were incubated with I0.5 nM 125 -EGF and increasing concentrations of cold rDIII (top) or EGF (bottom). The 0.5 nM (≈0.15 μCi) working concentration of I 125 -EGF was determined by calculating the specific binding of I 125 EGF (“specific”) based on total and nonspecific binding of I 125 -EGF to MDA-MB-231 cells (inset in bottom panel). Cells were incubated with increasing concentrations of I 125 -EGF in the absence (total binding; “total”) or presence of an excess amount (330 nM) of unlabeled EGF (nonspecific binding; “nonspecific”).

    Article Snippet: After 1 h on ice and washing, bound rDIII was stained with anti-His or 2778 antibody, followed either by fluorescently labeled anti–mouse or –rabbit antibody Alexa® 488 (IgG (H+L) F(ab')2 , Molecular Probes, Inc.).

    Techniques: Binding Assay, Flow Cytometry, Cytometry, Multiple Displacement Amplification, Incubation, Fluorescence, Concentration Assay