All lanes : Anti-RAGE antibody [EPR21171] (ab216329) at 1/2000 dilution

Lane 1 : Mouse lung lysates with 5% NFDM/TBST
Lane 2 : Mouse brain lysates with 5% NFDM/TBST
Lane 3 : Mouse kidney lysates with 5% NFDM/TBST
Lane 4 : Mouse heart lysates with 5% NFDM/TBST
Lane 5 : Mouse liver lysates with 5% NFDM/TBST
Lane 6 : Mouse spleen lysates with 5% NFDM/TBST
Lane 7 : Rat lung lysates with 5% NFDM/TBST
Lane 8 : Rat brain lysates with 5% NFDM/TBST
Lane 9 : Rat kidney lysates with 5% NFDM/TBST
Lane 10 : Rat heart lysates with 5% NFDM/TBST
Lane 11 : Rat spleen lysates with 5% NFDM/TBST

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 42 kDa
Observed band size: 43 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

The expression profile and molecular mass observed is consistent with what has been described in the literature (PMID:16315007; PMID:18355449; PMID:18245812)

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen rat lung tissue labeling RAGE with ab216329 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Positive membrane staining on alveolar epithelial cells, negative on the bronchial epithelial cells on rat lung tissue section is observed (PMID: 15173891).

The nuclear counter stain is DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

All lanes : Anti-RAGE antibody [EPR21171] (ab216329) at 1/1000 dilution

Lane 1 : Mouse lung lysate
Lane 2 : Rat lung lysate
Lane 3 : Human fetal lung lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lanes 1-2 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lane 3 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution

Developed using the ECL technique.

Predicted band size: 42 kDa
Observed band size: 45,55 kDa why is the actual band size different from the predicted?

Exposure times: Lane 1: 5 seconds; Lane 2: 10 seconds; Lane 3: 3 minutes.

Blocking/Dilution buffer: 5% NFDM/TBST.

The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument.

The expression profile and molecular mass observed is consistent with what has been described in the literature (PMID:16315007; PMID:18355449; PMID:18245812). Full-length RAGE is not detected in rat and human lysates.

Immunohistochemical analysis of paraffin-embedded human lung tissue labeling RAGE with ab216329 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous staining on epithelial cells of human lung (PMID: 19592063; PMID: 26472810) 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) Ready to use.

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

Immunohistochemical analysis of paraffin-embedded mouse lung tissue labeling RAGE with ab216329 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Mainly membranous staining on epithelial cells of mouse lung (PMID: 19592063; PMID: 26472810) 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) Ready to use.

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

Immunohistochemical analysis of paraffin-embedded rat lung tissue labeling RAGE with ab216329 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Mainly membranous staining on epithelial cells of rat lung (PMID: 19592063; PMID: 26472810) 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) Ready to use.

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

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen mouse lung tissue labeling RAGE with ab216329 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Positive membrane staining on alveolar epithelial cells, negative on the bronchial epithelial cells on mouse lung tissue section is observed (PMID: 15173891).

The nuclear counter stain is DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100-permeabilized HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells transfected with Myc-tagged RAGE expression vector labeling RAGE with ab216329 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing positive staining in HEK-293T cells transfected with Myc-tagged RAGE expression vector. 

The nuclear counter stain is DAPI (blue). Myc-Tag is detected with Myc-Tag (9B11) Mouse mAb (Alexa Fluor® 647 Conjugate) (red) at 1/1000 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Negative control: Myc-transfected HEK-293T cells.

Flow cytometric analysis of 4% paraformaldehyde-fixed HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells transfected with Myc-tagged RAGE expression vector labeling RAGE with ab216329 at 1/500 dilution (right panel) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (left panel). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Fresh cells were surface-stained with ab172730 and ab216329 respectively. Then fixed with 2% PFA for 15min and intracellular stained with anti-Myc tag antibody (Y axis). Only Myc+ population give positive signal.

RAGE was immunoprecipitated from 0.35 mg of mouse lung lysate with ab216329 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab216329 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution

Lane 1: Mouse lung lysate 10 µg (Input). 
Lane 2: ab216329 IP in mouse lung lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab216329 in mouse lung lysate.

 

Exposure time: 10 seconds.

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