Anti-GM130 抗体 [EP892Y] - cis-Golgi Marker

• IP

Unknown

Immunoprecipitation - Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (AB52649)

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

Lane 1 (input) : HeLa whole cell lysate (10μg)

Lane 2 (+) : ab52649 + HeLa whole cell lysate (10μg).

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab52649 in HeLa whole cell lysate.

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

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

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

Lane 1:

HeLa whole cell lysate at 10 µg

Lane 2:

ab52649 + HeLa whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab52649 in HeLa whole cell lysate

Predicted band size: 113 kDa

Observed band size: 130 kDa

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• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (AB52649)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - 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).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (AB52649)

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.

• ICC/IF

AbReview16956****

Immunocytochemistry/ Immunofluorescence - Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (AB52649)

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.

This image is courtesy of an Abreview submitted by Vladimir Milenkovic.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (AB52649)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (AB52649)

Intracellular 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 Fluorr^® 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.

• WB

Unknown

Western blot - Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (AB52649)

All lanes:

Western blot - Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (ab52649) at 1/200000 dilution

All lanes:

HeLa cell lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 113 kDa

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• WB

Lab

Western blot - Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (AB52649)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (ab52649) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 20 µg

Lane 2:

MCF7 cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 113 kDa

Observed band size: 130 kDa

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• WB

AbReview67200****

Western blot - Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (AB52649)

Blocking and dilution buffer : PBS, 0.05% Tween

Exposure : 5 minutes.

All lanes:

Western blot - Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (ab52649) at 1/1000 dilution

All lanes:

COS-7 Cell Line from African green monkey kidney whole cell lysate

Predicted band size: 113 kDa

false

• WB

Lab

Western blot - Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (AB52649)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (ab52649) at 1/5000 dilution

Lane 1:

MDCK(NBL-2) cell lysate at 20 µg

Lane 2:

MDCK(BL-1) cell lysate at 20 µg

Lane 3:

COS-1 cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 113 kDa

Observed band size: 130 kDa

false

• WB

AbReview20783****

Western blot - Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (AB52649)

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

Exposure : 10 minutes.

All lanes:

Western blot - Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (ab52649) at 1/2500 dilution

All lanes:

MDCK 2 cells at 25 µg

Predicted band size: 113 kDa

true