Recombinant Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (ab52649)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling GM130 with purified ab52649 at 1/50. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1: primary antibody (1/50) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

ab52649 (purified) at 1/20 immunoprecipitating GM130 in HeLa whole cell lysate.

For western blotting, ab131366 VeriBlot for IP (HRP) was used for detection (1/1500).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervix carcinoma tissue labelling GM130 with purified ab52649 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Blocking and dilution buffer: 5% NFDM/TBST.

Unpurified ab52649 staining GM130 in human ARPE-19 cells by ICC/IF (immunocytochemistry/immunofluorescence). Cells were formaldehyde fixed, permeabilized by 0.5% TX-100 and blocked with 5% serum for 20 minutes at 25°C. The sample was incubated with the primary antibody (1/500 in 1% goat serum, 0.1%TX100, 1 x PBS) for 16 hours at 4°C. An Alexa Fluor^® 488-conjugated Goat anti-rabbit polyclonal (1/500) was used as the secondary.

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ICC/IF image of unpurified ab52946 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (unpurified ab52946, 1µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight^® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Flow cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling GM130 (red) with ab52649 at a 1/20 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling GM130 with unpurified ab52649 at a dilution of 1/500.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Unpurified ab52649 staining GM130 (magenta) in monkey kidney cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and blocked with 3% BSA + 0.5% Triton X-100 for 45 minutes at 25°C. Samples were incubated with primary antibody (1/1500 in 3% BSA + 0.5% Triton X-100) for 45 minutes at 25°C. An Alexa Fluor^® 647-conjugated donkey anti-rabbit IgG polyclonal (2 µg/ml) was used as the secondary antibody. Nuclei stained with Picogreen.

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Blocking and dilution buffer: 5% NFDM/TBST.

Unpurified ab52649 staining GM130 in Bovine brain microvascular endothelial cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.1% saponin and blocked with 5% BSA for 90 minutes at 37°C. Samples were incubated with primary antibody (1/100 in 0.1% saponin + 1% BSA ) for 18 hours at 4°C. An undiluted Alexa Fluor^® 568-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.

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Immunocytochemistry/Immunofluorescence analysis of MDCK II cells grown on the filter labelling GM130 with purified ab52649 at 1/1000. Cells were fixed with 5% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. 

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Blocking and dilution buffer: 1XPBS-Tween, 5% milk

Exposure: 10 minutes.

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Immunocytochemistry/Immunofluorescence analysis of COS7 (African green monkey) labelling GM130 with purified ab52649 with concentration of 10µg/ml. Cells were fixed with 5% paraformaldehyde and permeabilized with 0.2% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

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Blocking and dilution buffer: PBS, 0.05% Tween 

Exposure: 5 minutes.

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