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.

All lanes :

Lane 1 : HeLa whole cell lysate at 10 µg
Lane 2 : ab52649 + HeLa whole cell lysate at 10 µg
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab52649 in HeLa whole cell lysate

Observed band size: 130 kDa why is the actual band size different from the predicted?

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.

All lanes : Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (ab52649) at 1/1000 dilution (purified)

Lane 1 : HeLa cell lysate
Lane 2 : MCF7 cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 112 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?

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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Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (ab52649) at 1/200000 dilution (unpurified) + HeLa cell lysate at 10 µg

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

Predicted band size: 112 kDa

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.

All lanes : Anti-GM130 antibody [EP892Y] - cis-Golgi Marker (ab52649) at 1/5000 dilution (purified)

Lane 1 : MDCK(NBL-2) cell lysate
Lane 2 : MDCK(BL-1) cell lysate
Lane 3 : COS-1 cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 112 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

MDCK 2 cells at 25 µg

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 112 kDa

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

Exposure: 10 minutes.

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

Predicted band size: 112 kDa

Blocking and dilution buffer: PBS, 0.05% Tween 

Exposure: 5 minutes.

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