Anti-Ras (mutated Q61R) antibody [SP174]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ras (mutated Q61R) antibody [SP174] (AB227658)

IHC image of NRAS (mutated Q61R) staining in a section of formalin-fixed paraffin-embedded human skin melanoma* performed on a Leica BONDTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with EDTA buffer (pH9, epitope retrieval solution 2) for 20mins. The section was then incubated with ab227658, 1/100 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Ras (mutated Q61R) antibody [SP174] (AB227658)

Flow Cytometry analysis of SK-MEL-2( Human skin malignant melanoma) cells labeling NRAS with purifiedab227658 at 1 : 200 dilution (0.8 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 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / blue.

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ras (mutated Q61R) antibody [SP174] (AB227658)

Immunohistochemical analysis of paraffin-embedded human primary malignant melanoma tissue (mutation status : NRAS Q61R) labeling NRAS with ab227658 at 1/100 dilution for 30 min at RT. The tissue was counterstained with Hematoxylin. Heat mediated antigen retrieval was achieved by using EDTA buffer pH 9.0 before IHC staining.

This image is courtesy of an anonymous collaborator.

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ras (mutated Q61R) antibody [SP174] (AB227658)

Immunohistochemical analysis of paraffin-embedded human primary malignant melanoma tissue (mutation status : NRAS Q61R) labeling NRAS with ab227658 at 1/100 dilution for 30 min at RT. The tissue was counterstained with Hematoxylin. Heat mediated antigen retrieval was achieved by using EDTA buffer pH 9.0 before IHC staining.

This image is courtesy of an anonymous collaborator.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ras (mutated Q61R) antibody [SP174] (AB227658)

Immunohistochemistry analysis of paraffin-embedded (A) HEK-293T transfected with NRAS (Q61R) expression vector, (B) HEK-293T transfected with NRAS (Q61K) expression vector, (C) HEK-293T transfected with NRAS (Q61H) expression vector, (D) HEK-293T transfected with NRAS (Q61L) expression vector, (E) HEK-293T transfected with NRAS expression vector, (F) HEK-293T transfected with HRAS (Q61R) expression vector, (G) HEK-293T transfected with KRAS(Q61R) and (H) HEK-293T transfected empty vector, sections labelling NRAS (mutated Q61R) with ab227658 at 1/100 dilution. The section was incubated with ab227658 for 10 mins at room temperature. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Sections were counterstained with Hematoxylin. Antigen retrieval was heat mediated using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 20 minutes. Positive staining on HEK-293T transfected with NRAS (Q61R) expression vector (Image A), HEK-293T transfected with NRAS (Q61K) expression vector (Image B), HEK-293T transfected with HRAS (Q61R) expression vector (Image F) and HEK-293T transfected with KRAS (Q61R) expression vector (Image G); Nearly no staining on HEK-293T transfected with NRAS (Q61H) expression vector (Image C), HEK-293T transfected with NRAS (Q61L) expression vector (Image D), HEK-293T transfected with NRAS expression vector (Image E) and HEK-293T transfected empty vector (Image H). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ras (mutated Q61R) antibody [SP174] (AB227658)

Immunohistochemical analysis of paraffin-embedded human metastatic malignant melanoma tissue (mutation status : NRAS Q61K) labeling NRAS with ab227658 at 1/100 dilution for 30 min at RT. The tissue was counterstained with Hematoxylin. Heat mediated antigen retrieval was achieved by using EDTA buffer pH 9.0 before IHC staining.

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ras (mutated Q61R) antibody [SP174] (AB227658)

Immunohistochemical analysis of paraffin-embedded human primary malignant melanoma tissue (mutation status : NRAS Q61R) labeling NRAS with ab227658 at 1/100 dilution for 30 min at RT. The tissue was counterstained with Hematoxylin. Heat mediated antigen retrieval was achieved by using EDTA buffer pH 9.0 before IHC staining.

This image is courtesy of an anonymous collaborator.

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ras (mutated Q61R) antibody [SP174] (AB227658)

Immunohistochemical analysis of paraffin-embedded human metastatic malignant melanoma tissue (mutation status : NRAS Q61R) labeling NRAS with ab227658 at 1/100 dilution for 30 min at RT. The tissue was counterstained with Hematoxylin. Heat mediated antigen retrieval was achieved by using EDTA buffer pH 9.0 before IHC staining.

This image is courtesy of an anonymous collaborator.

• WB

Lab

Western blot - Anti-Ras (mutated Q61R) antibody [SP174] (AB227658)

Blocking buffer and concentration : 5% NFDM/TBST Diluting buffer and concentration : 5% NFDM /TBST This antibody has been found to react with different mutations we tested. We haven’t tested all possible mutations at Q61.

All lanes:

Western blot - Anti-Ras (mutated Q61R) antibody [SP174] (ab227658) at 1/1000 dilution

Lane 1:

HEK-293T (Human embryonic kidney epithelial cell) transfected with an empty vector (vector control), containing a His-tag, whole cell lysate at 20 µg

Lane 2:

HEK-293T (Human embryonic kidney epithelial cell) transfected with His-tagged NRAS (mutated Q61R) whole cell lysate at 20 µg

Lane 3:

HEK-293T (Human embryonic kidney epithelial cell) transfected with His-tagged NRAS (mutated Q61K) whole cell lysate at 20 µg

Lane 4:

HEK-293T (Human embryonic kidney epithelial cell) transfected with His-tagged NRAS (mutated Q61H) whole cell lysate at 20 µg

Lane 5:

HEK-293T (Human embryonic kidney epithelial cell) transfected with His-tagged NRAS (mutated Q61L) whole cell lysate at 20 µg

Lane 6:

HEK-293T (Human embryonic kidney epithelial cell) transfected with His-tagged NRAS whole cell lysate at 20 µg

Lane 7:

HEK-293T (Human embryonic kidney epithelial cell) transfected with His-tagged HRAS (mutated Q61R) whole cell lysate at 20 µg

Lane 8:

HEK-293T (Human embryonic kidney epithelial cell) transfected with His-tagged KRAS (mutated Q61R) whole cell 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 1/20000 dilution

Predicted band size: 21 kDa

Observed band size: 25 kDa

false

Exposure time: 5s

• WB

Supplier Data

Western blot - Anti-Ras (mutated Q61R) antibody [SP174] (AB227658)

All lanes:

Western blot - Anti-Ras (mutated Q61R) antibody [SP174] (ab227658) at 1/400 dilution

All lanes:

SK-MEL-2 cell lysate

Predicted band size: 21 kDa

false