Anti-ROR gamma antibody [EPR20006]

• Flow Cyt (Intra)

Lab

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

Flow cytometry overlay histogram showing mouse thymocytes stained with ab207082 (right) and isotype control (left).

The cells were incubated in 1x PBS containing 10μg/ml anti CD16/CD32 and 10% normal goat serum to block FC receptors and non-specific protein-protein interactions. The cells were then fixed and permeabilised with CytoFix/CytoPerm, followed by addition of the antibody (ab207082) (1x 10⁶ in 100μl at 1.0μg/ml (1/2,000)) for 30min at 22°C.

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

Acquisition of >30,000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on live cells.

• 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='/ko/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='/ko/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