Anti-TGN46 antibody [EPR24770-16] - Golgi Marker

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-TGN46 antibody [EPR24770-16] - Golgi Marker (AB271183)

Flow cytometry overlay histogram showing wild-type A549 (green line) and TGOLN2 knockout A549 stained with ab271183 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab271183) (1x 106 in 100μl at 1.0 μg/ml (1/1980)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type A549 - black line, TGOLN2 knockout A549 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in A549 Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TGN46 antibody [EPR24770-16] - Golgi Marker (AB271183)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labelling TGN46 with ab271183 at 1/2000 (0.297 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining in human cerebrum. The section was incubated with ab271183 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TGN46 antibody [EPR24770-16] - Golgi Marker (AB271183)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labelling TGN46 with ab271183 at 1/2000 (0.297 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining in human kidney. The section was incubated with ab271183 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TGN46 antibody [EPR24770-16] - Golgi Marker (AB271183)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling TGN46 with ab271183 at 1/50 dilution (1 μg)/ (Red), compared with a Rabbit monoclonal IgG (ab172730)/ (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TGN46 antibody [EPR24770-16] - Golgi Marker (AB271183)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labelling TGN46 with ab271183 at 1/2000 (0.297 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining in human colon. The section was incubated with ab271183 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-TGN46 antibody [EPR24770-16] - Golgi Marker (AB271183)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 cells labelling TGN46 with ab271183 at 1/50 (11.88 μg/mL) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 μg/mL) dilution (Green). Confocal image showing co-locolization staining with golgi marker in HepG2 cells is observed. ab169276 Anti-GM130 mouse polyclonal antibody - cis-Golgi Marker was used to counterstain tubulin at 1/200 (5 μg/mL) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Negative Control 1 : Secondary antibody only control : ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 μg/mL) dilution.

Negative Control 2 : ab169276 Anti-GM130 mouse polyclonal antibody - cis-Golgi Marker 1/200 (5 μg/ml) and Secondary antibody ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 μg/mL) dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TGN46 antibody [EPR24770-16] - Golgi Marker (AB271183)

Immunohistochemical analysis of paraffin-embedded Human lung carcinoma tissue labelling TGN46 with ab271183 at 1/2000 (0.297 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining in human lung carcinoma. The section was incubated with ab271183 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IP

Supplier Data

Immunoprecipitation - Anti-TGN46 antibody [EPR24770-16] - Golgi Marker (AB271183)

TGN46 was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate 10 ug with ab271183 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab271183 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate 10 μg

Lane 2 : HepG2 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab271183 in HepG2 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 50 seconds

Lysates were made freshly and used in IP immediately to minimize protein degradation. Lower bands observed in lane 2 may caused by degradation during incubation based on the WB results.

All lanes:

Immunoprecipitation - Anti-TGN46 antibody [EPR24770-16] - Golgi Marker (ab271183)

Predicted band size: 51 kDa

Observed band size: 80-100 kDa

false

• WB

Lab

Western blot - Anti-TGN46 antibody [EPR24770-16] - Golgi Marker (AB271183)

Blocking and diluting buffer and concentration : 3% NFDM/TBST.

Lanes 1-3 : Merged signal (red and green).

Green : ab271183 observed at 80-100 kDa.

Red : loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.

Lanes 1-2 : ab271183 was shown to react with TGN46 in wild-type A549 cells in Western blot with loss of signal observed in TGN46 knockout cell line ab269510 (knockout cell lysate ab269672). Wild-type A549 and TGN46 knockout cell lysates were subjected to SDS-PAGE.

ab271183 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-TGN46 antibody [EPR24770-16] - Golgi Marker (ab271183) at 1/1000 dilution

Lane 1:

Wild-type A549 (human lung carcinoma epithelial cell) whole cell lysate at 40 µg

Lane 2:

TGOLN2 (TGN46) knockout A549 (human lung carcinoma epithelial cell) whole cell lysate at 40 µg

Lane 2:

Western blot - Human TGOLN2 (TGN46) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-tgoln2-tgn46-knockout-a549-cell-line-ab269510'>ab269510</a>)

Lane 3:

HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at a 1/10000 dilution.

Predicted band size: 51 kDa

Observed band size: 80-100 kDa

false

• WB

Supplier Data

Western blot - Anti-TGN46 antibody [EPR24770-16] - Golgi Marker (AB271183)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID : 15616480; PMID : 22909819).

Lysates were made freshly and used in WB immediately to minimize protein degradation.

Exposure time : Lane 1 : 26 seconds; Lane 2 : 125 seconds.

All lanes:

Western blot - Anti-TGN46 antibody [EPR24770-16] - Golgi Marker (ab271183) at 1/1000 dilution

Lane 1:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 51 kDa

Observed band size: 80-100 kDa

false