Anti-Clathrin antibody [X22]

• Supplier: Abcam
• Catalog: ab2731
• Prices: $403.00
• Sizes: 100 µl
• Host: Mouse
• Clonality: Monoclonal
• Isotype: IgG1
• Clone: X22
• Applications: ICC/IF ICC/IF ICC/IF ELISA B/N FC Immunomicroscopy IP WB IHC-P
• Species Reactivities: Mouse, Rat, Bovine, Dog, Human, Pig, Xenopus, Bird, Monkey
• Antigen: Full length native protein (purified) corresponding to Human Clathrin
• Description: Mouse Monoclonal
• Gene: AP1B1
• Conjugate: Unconjugated

Product images

Immunocytochemistry/Immunofluorescence analysis of Clathrin shows staining in HeLa cells. Clathrin, Heavy chain staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with ab2731 (1:200) over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-mouse secondary antibody. Images were taken at 60X magnification.

Immunocytochemistry/Immunofluorescence analysis of Clathrin shows staining in NCI-H460 cells. Clathrin, Heavy chain staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with ab2731 (1:200) over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-mouse secondary antibody. Images were taken at 60X magnification.

Immunocytochemistry/Immunofluorescence analysis of Clathrin shows staining in U251 cells. Clathrin, Heavy chain staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with ab2731 (1:200) over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-mouse secondary antibody. Images were taken at 60X magnification.

ab2731 staining human HEK 293 cells by ICC/IF.  Cells were PFA fixed and permeabilized in 0.01% Triton X-100 in PBS prior to blocking in 1% BSA for 1 hour at 22°C.  The primary antibody was diluted 1/75 and incubated with the sample for 1 hour at 22°C.  An Alexa Fluor® 405 conjugated goat anti-mouse antibody diluted 1/400 was used as the secondary.See Abreview

All lanes : Anti-Clathrin antibody [X22] (ab2731) at 1/500 dilutionLane 1 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate Lane 2 : Raji (Human Burkitt's lymphoma cell line) Whole Cell Lysate Lysates/proteins at 10 µg per lane.SecondaryGoat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilutionObserved band size : 180 kDa (why is the actual band size different from the predicted?)Additional bands at : 240 kDa,450 kDa. We are unsure as to the identity of these extra bands.

ab2731 staining Clathrin - Membrane Vesicle Marker in Rat hypothalamus primary cells by ICC/IF (immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.5% TX-100 and blocked with 5% serum for 20 minutes at 25°C. Samples were incubated with primary antibody (1/500 in 1% goat serum; 0.1% TX100; PBS) for 16 hours at 4°C. An Alexa Fluor®546-conjugated Goat polyclonal to mouse IgG, dilution 1/500, was used as secondary antibody. See Abreview

ab2731 staining Clathrin in pig retinal pigment epithelium (RPE) cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed with paraformaldehyde, permeabilized with 0.5% Triton ×100 and blocked with 5% serum for 20 minutes at 250C. Samples were incubated with primary antibody (1/500: in 0.1% TX100, 1% goat serum, 1X PBS) for 16 hours at 4°C. An Alexa Fluor®546-conjugated goat polyclonal to mouse IgG was used as secondary antibody at 1/500 dilution.See Abreview

ab2731 staining clathrin in Human platelets by Flow Cytometry. Platelets were isolated form Platelet rich plasma and suspended in PBS, fixed with paraformaldehyde and permeabilized with 0.1% Triton-X100 in 2% BSA for 30 minutes. The sample was incubated with the primary antibody (1/200 in PBS + 2% BSA) for 18 hours at 4°C. An Alexa Fluor®594-conjugated Goat anti-mouse IgG polyclonal (1/500) was used as the secondary antibody. Gating Strategy: PlateletsSee Abreview

Immunohistochemistry was performed on cancer biopsies of deparaffinized Human breast carcinoma tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing Clathrin Heavy chain ab2731 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Immunohistochemistry was performed on normal biopsies of deparaffinized Human colon tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing Clathrin Heavy chain ab2731 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Xenopus laevis cytoplasmic egg extract visualized live with primary and secondary antibody addition [red is anti-Clathrin X22 (ab2731) with goat anti-mouse Alexa Fluor 568 secondary, green is anti-HIP1R (ab77297) with goat anti-rabbit Alexa Fluor 488 secondary]. Large red structures are probably aggregates, but the small structures appear to be specific for vesicle staining.See Abreview

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