Anti-ROR gamma antibody [EPR20006]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ROR gamma antibody [EPR20006] (AB207082)

Flow cytometry overlay histogram showing positive mouse thymocytes on the right and negative mouse thymocytes on the left, both stained with ab207082 (red line). The cells were fixed with Cytofix and then permeabilised with CytoPerm. The cells were then incubated in 1x PBS containing 10μg/ml anti CD16/CD32 and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab207082) (1x 10⁶ in 100μl at 1.0μg/ml (1/2,000 dilution)) for 30 mins at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/2000 dilution for 30 mins at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on CD8+ve.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ROR gamma antibody [EPR20006] (AB207082)

Immunohistochemical analysis of paraffin-embedded mouse thymus tissue labeling ROR gamma with ab207082 at 1/3000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear staining on the cortical region of mouse thymus shown at a high magnification (400×) (PMID : 14691482).

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ROR gamma antibody [EPR20006] (AB207082)

Immunohistochemical analysis of paraffin-embedded mouse thymus tissue labeling ROR gamma with ab207082 at 1/3000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear staining on mouse thymus cortex, located in the left and right of the image, with sporadic nuclear staining on medulla, middle region (PMID : 14691482).

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ROR gamma antibody [EPR20006] (AB207082)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling ROR gamma with ab207082 at 1/3000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Sporadic nuclear staining on mouse spleen (PMID : 14691482).

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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-ROR gamma antibody [EPR20006] (AB207082)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed Mouse thymocytes labeling ROR gamma with ab207082 at 1/50 dilution. Goat anti rabbit IgG (Alexa Fluorr^® 488) at 1/2000 dilution was used as the secondary antibody.

Note : Cell surface staining with PE conjugated anti-mouse CD8 and Alexa Fluorr^® 647 conjugated anti mouse CD4 antibodies, followed by fixation with 4% PFA for 10 min and intracellular staining with ab207082. The image shows thymocytes in the CD4+CD8+ (red) or the CD4+CD8- (black, negative control) gate.

• IP

Supplier Data

Immunoprecipitation - Anti-ROR gamma antibody [EPR20006] (AB207082)

ROR gamma was immunoprecipitated from 0.35 mg of Mouse thymus lysate with ab207082 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab207082 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : Mouse thymus lysate, 10 μg (Input).

Lane 2 : ab207082 IP in Mouse thymus lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab207082 inMouse thymus lysate.

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

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-ROR gamma antibody [EPR20006] (ab207082)

Predicted band size: 58 kDa

Observed band size: 58 kDa

false

• WB

Supplier Data

Western blot - Anti-ROR gamma antibody [EPR20006] (AB207082)

Blocking/Dilution buffer : 5% NFDM/TBST.

The expression profile is consistent with the literature : PMID 14691482; PMID 9881970.

All lanes:

Western blot - Anti-ROR gamma antibody [EPR20006] (ab207082) at 1/2000 dilution

Lane 1:

Mouse thymus lysate at 20 µg

Lane 2:

Mouse liver 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 1/100000 dilution

Predicted band size: 58 kDa

Observed band size: 58 kDa

false

Exposure time: 3s

• WB

Supplier Data

Western blot - Anti-ROR gamma antibody [EPR20006] (AB207082)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 3 seconds; Lane 2/3 : 3 minutes.

Lane 1:

Western blot - Anti-ROR gamma antibody [EPR20006] (ab207082) at 1/10000 dilution

Lanes 2 - 3:

Western blot - Anti-ROR gamma antibody [EPR20006] (ab207082) at 1/1000 dilution

Lane 1:

His-tagged mouse ROR gamma recombinant protein (aa 1-266) at 0.01 µg

Lane 2:

His-tagged mouse ROR alpha recombinant protein (aa 48-215) at 0.01 µg

Lane 3:

His-tagged mouse ROR beta recombinant protein (aa 9-140) at 0.01 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 1/100000 dilution

Predicted band size: 58 kDa

Observed band size: 35 kDa

false