All lanes : Anti-Ionotropic Glutamate receptor 2 antibody [EPR18115] (ab206293) at 1/5000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Rat brain lysate
Lane 3 : Human fetal brain lysate
Lane 4 : Rat spleen lysate
Lane 5 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 6 : Human fetal kidney lysate
Lane 7 : HT1080 (Human fibrosarcoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 99 kDa
Observed band size: 99 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 1 second; Lane 2,3,4,5,6 and 7: 10 seconds.

All lanes : Anti-Ionotropic Glutamate receptor 2 antibody [EPR18115] (ab206293) at 1/2000 dilution

Lane 1 : Human brain lysate
Lane 2 : Mouse brain lysate
Lane 3 : Rat brain lysate
Lane 4 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 5 : Human fetal kidney lysate
Lane 6 : HT1080 (Human fibrosarcoma cell line) whole cell lysate
Lane 7 : Mouse heart lysate
Lane 8 : Rat spleen lysate
Lane 9 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 99 kDa
Observed band size: 99 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human solitary fibrous tumor tissue labeling Ionotropic Glutamate receptor 2 with ab206293 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Membrane staining on Human solitary fibrous tumor is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HT1080 (Human fibrosarcoma cell line) cells labeling Ionotropic Glutamate receptor 2 with ab206293 at 1/700 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

 

Confocal image showing cytoplasmic staining on HT1080 cell line.

 

The nuclear counterstain is DAPI (blue).

 

Tubulin is detected with Anti-alpha Tubulin antibody [EPR18115] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

 

The negative controls are as follows:-

-ve control 1: ab206293 at 1/700 dilution followed by ab150120  at 1/1000 dilution.

-ve control 2: ab7291  at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

Ionotropic Glutamate receptor 2 was immunoprecipitated from 1mg of C6 (Rat glial tumor cell line) whole cell lysate with ab206293 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab206293 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: C6 whole cell lysate 10µg (Input).

Lane 2: ab206293 IP in C6 whole cell lysate.

Lane 3:Rabbit IgG, monoclonal [EPR18115] - Isotype Control (ab172730) instead of ab206293 in C6 whole cell lysate.

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

Exposure time: 10 seconds.