Anti-RHEB antibody [EPR2971]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-RHEB antibody [EPR2971] (AB92313)

Immunocytochemistry/ Immunofluorescence analysis of Raji (Human Burkitt's lymphoma B lymphocyte) cells labeling RHEB with purified ab92313 at 1 : 250 dilution (1.46 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-RHEB antibody [EPR2971] (AB92313)

Intracellular Flow Cytometry analysis of Raji (Human Burkitt's lymphoma B lymphocyte) cells labeling RHEB with purified ab92313 at 1/40 dilution (10 μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RHEB antibody [EPR2971] (AB92313)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human glioma tissue sections labeling RHEB with purified ab92313 at 1 : 500 dilution (0.73 µg/ml). Heat mediated antigen retrieval was performed using Bond ™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-RHEB antibody [EPR2971] (AB92313)

Overlay histogram showing Raji cells stained with unpurifiedab92313 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab92313, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in Raji cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RHEB antibody [EPR2971] (AB92313)

Unpurified ab92313, at 1/100 dilution, staining RHEB in paraffin-embedded Human brain tissue, by Immunohistochemistry.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• WB

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Western blot - Anti-RHEB antibody [EPR2971] (AB92313)

All lanes:

Western blot - Anti-RHEB antibody [EPR2971] (ab92313) at 1/2000 dilution

Lane 1:

Raji cell lsyate at 10 µg

Lane 2:

SH-SY5Y cell lsyate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 20 kDa

false

• WB

Lab

Western blot - Anti-RHEB antibody [EPR2971] (AB92313)

All lanes:

Western blot - Anti-RHEB antibody [EPR2971] (ab92313) at 1/2000 dilution

All lanes:

Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysates at 15 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: 20 kDa

false