Anti-RAGE antibody [EPR21171] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAGE antibody [EPR21171] - BSA and Azide free (AB228861)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216329).

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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RAGE antibody [EPR21171] - BSA and Azide free (AB228861)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216329).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAGE antibody [EPR21171] - BSA and Azide free (AB228861)

This data was developed using ab216329, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of formalin fixed paraffin embedded human lung labelling RAGE with ab216329 at a concentration of 0.05µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab216329 anti-RAGE antibody [EPR21171] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• Flow Cyt

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Flow Cytometry - Anti-RAGE antibody [EPR21171] - BSA and Azide free (AB228861)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216329).

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-RAGE antibody [EPR21171] - BSA and Azide free (AB228861)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216329).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAGE antibody [EPR21171] - BSA and Azide free (AB228861)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216329).

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAGE antibody [EPR21171] - BSA and Azide free (AB228861)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216329).

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

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-RAGE antibody [EPR21171] - BSA and Azide free (AB228861)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216329).

• IP

Supplier Data

Immunoprecipitation - Anti-RAGE antibody [EPR21171] - BSA and Azide free (AB228861)

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 1/10000 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab216329).

All lanes:

Immunoprecipitation - Anti-RAGE antibody [EPR21171] (<a href='/en-us/products/primary-antibodies/rage-antibody-epr21171-ab216329'>ab216329</a>)

Predicted band size: 42 kDa

Observed band size: 45 kDa,55 kDa

true

• WB

Supplier Data

Western blot - Anti-RAGE antibody [EPR21171] - BSA and Azide free (AB228861)

This data was developed using ab216329, the same antibody clone in a different buffer formulation.

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.

All lanes:

Western blot - Anti-RAGE antibody [EPR21171] (<a href='/en-us/products/primary-antibodies/rage-antibody-epr21171-ab216329'>ab216329</a>) at 1/1000 dilution

Lane 1:

Mouse lung lysate at 10 µg

Lane 2:

Rat lung lysate at 10 µg

Lane 3:

Human fetal lung lysate at 10 µg

Secondary

Lanes 1 - 2:

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 1/100000 dilution

Lane 3:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/4000 dilution

Predicted band size: 42 kDa

Observed band size: 45 kDa,55 kDa

true

• WB

Supplier Data

Western blot - Anti-RAGE antibody [EPR21171] - BSA and Azide free (AB228861)

This data was developed using ab216329, the same antibody clone in a different buffer formulation.

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

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

All lanes:

Western blot - Anti-RAGE antibody [EPR21171] (<a href='/en-us/products/primary-antibodies/rage-antibody-epr21171-ab216329'>ab216329</a>) at 1/2000 dilution

Lane 1:

Mouse lung lysates at 20 µg

Lane 2:

Mouse brain lysates at 20 µg

Lane 3:

Mouse kidney lysates at 20 µg

Lane 4:

Mouse heart lysates at 20 µg

Lane 5:

Mouse liver lysates at 20 µg

Lane 6:

Mouse spleen lysates at 20 µg

Lane 7:

Rat lung lysates at 20 µg

Lane 8:

Rat brain lysates at 20 µg

Lane 9:

Rat kidney lysates at 20 µg

Lane 10:

Rat heart lysates at 20 µg

Lane 11:

Rat spleen lysates 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 1/20000 dilution

Predicted band size: 42 kDa

Observed band size: 43 kDa

false

Exposure time: 3s