Name: Anti-Ras antibody [EPR23474-20]
SKU: ab275875
Rating: 5 (4 reviews)

Rabbit Recombinant Monoclonal RASK antibody. Suitable for mIHC, IP, WB, Flow Cyt (Intra), IHC-P and reacts with Mouse, Human, Rat, Recombinant full length protein - Human samples.

Images

Immunoprecipitation - Anti-Ras antibody [EPR23474-20] (AB275875), expandable thumbnail

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: Preservative: 0.01% Sodium azide
Constituents: 59.94% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	mIHC	ICC/IF	IP	WB	Flow Cyt (Intra)	IHC-P
Human	Expected	Not recommended	Tested	Tested	Tested	Tested
Mouse	Tested	Not recommended	Not recommended	Tested	Tested	Tested
Rat	Expected	Not recommended	Expected	Tested	Expected	Tested
Recombinant full length protein - Human	Not recommended	Not recommended	Not recommended	Tested	Not recommended	Not recommended

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/2000	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Expected
Expected

Species	Dilution info	Notes
Species Human, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant full length protein - Human	Dilution info -	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Mouse	Dilution info -	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Rat	Dilution info -	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Human	Dilution info -	Notes -
Species Recombinant full length protein - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/30	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Rat	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Mouse, Recombinant full length protein - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Recombinant full length protein - Human	Dilution info 1/1000	Notes -
Species Mouse	Dilution info 1/1000	Notes -
Species Rat	Dilution info 1/1000	Notes -
Species Human	Dilution info 1/1000	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/50	Notes -
Species Human	Dilution info 1/50	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Rat	Dilution info Use at an assay dependent concentration.	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant full length protein - Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/2000	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Rat	Dilution info 1/2000	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Human	Dilution info 1/2000	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Not recommended
Not recommended

Species	Dilution info	Notes
Species Recombinant full length protein - Human	Dilution info -	Notes -

Associated Products

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2 products for Alternative Product

Target data

See full target information KRAS

Function

The protein expressed by the KRAS gene binds GDP/GTP and has intrinsic GTPase activity. It plays an important role in regulating cell proliferation and may contribute to oncogenic events by inducing transcriptional silencing of tumor suppressor genes in colorectal cancer cells through a ZNF304-dependent mechanism. This supplementary information is collated from multiple sources and compiled automatically.

Alternative names

KRAS2, RASK2, KRAS, GTPase KRas, K-Ras 2, Ki-Ras, c-K-ras, c-Ki-ras

Recommended products

Rabbit Recombinant Monoclonal RASK antibody. Suitable for mIHC, IP, WB, Flow Cyt (Intra), IHC-P and reacts with Mouse, Human, Rat, Recombinant full length protein - Human samples.

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: Preservative: 0.01% Sodium azide
Constituents: 59.94% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Clone number: EPR23474-20
Purification technique: Affinity purification Protein A
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

This product is a recombinant monoclonal antibody, which offers several advantages including:

- High batch-to-batch consistency and reproducibility
- Improved sensitivity and specificity
- Long-term security of supply
- Animal-free batch production

For more information, read more on recombinant antibodies.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

The Ras protein also known by its alternate names such as KRAS NRAS and HRAS functions as a small GTPase which is a type of enzyme that binds to guanosine triphosphate (GTP) and can switch to an inactive form when it hydrolyzes GTP to GDP. Ras proteins are encoded by genes located in different chromosomes and typically have a molecular weight of about 21 kDa. These proteins are expressed in various tissues throughout the body where they play key roles in cellular signaling.

Biological function summary

Ras proteins are pivotal in regulating cell proliferation differentiation and survival. They are often part of larger protein complexes that facilitate signal transduction across cell membranes. These proteins function as binary molecular switches toggling between active (GTP-bound) and inactive (GDP-bound) states. Mutations in Ras proteins such as NRAS Q61R can lead the protein to assume permanently active conformations disrupting normal cellular signaling processes and contributing to oncogenesis.

Pathways

Ras proteins play significant roles in the MAPK/ERK and PI3K/AKT signaling pathways. Their activation leads to a cascade of phosphorylation events that ultimately regulate gene expression. Within these pathways Ras proteins interact with various molecules including RAF kinases and the PI3K protein further illustrating their complex roles in signal propagation. These pathways control many cellular processes including growth differentiation and survival illustrating how Ras proteins integrate multiple signals to modulate cellular outcomes.

Associated diseases and disorders

Ras proteins are frequently implicated in various cancers notably pancreatic and colorectal cancers. Mutated forms of Ras such as KRAS G12V drive oncogenesis by promoting uncontrolled cell growth and division. In cancer pathology Ras often interacts with tumor suppressor proteins influencing the disease progression. Targeting Ras-mediated signaling pathways using approaches like pan-Ras inhibitors or chemiluminescence ELISAs represents an ongoing area of therapeutic research aimed at combating Ras-driven malignancies.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

17 product images

Immunoprecipitation - Anti-Ras antibody [EPR23474-20] (ab275875)
Ras was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab275875 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab275875 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug
Lane 2: ab275875 IP in HeLa whole cell lysate
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab275875 in HeLa whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 110 seconds.
All lanes: Immunoprecipitation - Anti-Ras antibody [EPR23474-20] (ab275875)
Predicted band size: 21 kDa
Observed band size: 21 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ras antibody [EPR23474-20] (ab275875)
Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Ras with ab275875 at 1/2000 (0.338 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Positive staining on human colon. The section was incubated with ab275875 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Immunoprecipitation - Anti-Ras antibody [EPR23474-20] (ab275875)
Ras was immunoprecipitated from 0.35 mg A431 (human epidermoid carcinoma epithelial cell) whole cell lysate 10 with ab275875 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab275875 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
Lane 1: A431 (human epidermoid carcinoma epithelial cell) whole cell lysate 10 ug
Lane 2: ab275875 IP in A431 whole cell lysate
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab275875 in A431 whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 50 seconds.
All lanes: Immunoprecipitation - Anti-Ras antibody [EPR23474-20] (ab275875)
Predicted band size: 21 kDa
Observed band size: 21 kDa
Western blot - Anti-Ras antibody [EPR23474-20] (ab275875)
Blocking and diluting buffer and concentration: 5% NFDM/TBST
Exposure time: 26 seconds.
All lanes: Western blot - Anti-Ras antibody [EPR23474-20] (ab275875) at 1/1000 dilution
Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 40 µg
Lane 2: HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 40 µg
Lane 3: A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 40 µg
Lane 4: Human brain tissue lysate at 40 µg
Secondary
All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/50000 dilution
Predicted band size: 21 kDa
Observed band size: 21 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ras antibody [EPR23474-20] (ab275875)
Immunohistochemical analysis of paraffin-embedded Mouse stomach tissue labeling Ras with ab275875 at 1/2000 (0.338 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Positive staining on Mouse stomach. The section was incubated with ab275875 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Western blot - Anti-Ras antibody [EPR23474-20] (ab275875)
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
Exposure time: Lanes 1-2: 26 seconds; Lane3: 10 seconds.
All lanes: Western blot - Anti-Ras antibody [EPR23474-20] (ab275875) at 1/1000 dilution
Lane 1: Human colon tissue lysate at 40 µg
Lane 2: Human liver tissue lysate at 40 µg
Lane 3: Human stomach tissue lysate at 40 µg
Secondary
All lanes: VeriBlot for IP secondary antibody(HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/1000 dilution
Predicted band size: 21 kDa
Observed band size: 21 kDa
Western blot - Anti-Ras antibody [EPR23474-20] (ab275875)
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
Exposure time: Lanes 1-3: 26 seconds; Lane 4: 70 seconds.
All lanes: Western blot - Anti-Ras antibody [EPR23474-20] (ab275875) at 1/1000 dilution
Lane 1: C6 (rat glial tumor glial cell) whole cell lysate at 20 µg
Lane 2: RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg
Lane 3: PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg
Lane 4: NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg
Secondary
All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/50000 dilution
Predicted band size: 21 kDa
Observed band size: 21 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ras antibody [EPR23474-20] (ab275875)
Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling Ras with abab275875 at 1/2000 (0.338 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Positive staining on rat colon. The section was incubated with ab275875 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Western blot - Anti-Ras antibody [EPR23474-20] (ab275875)
Blocking and diluting buffer and concentration: 5% NFDM/TBST
Exposure time: 3 seconds.
All lanes: Western blot - Anti-Ras antibody [EPR23474-20] (ab275875) at 1/1000 dilution
Lane 1: Mouse colon tissue lysate at 40 µg
Lane 2: Mouse stomach tissue lysate at 40 µg
Lane 3: Rat colon tissue lysate at 40 µg
Secondary
All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/50000 dilution
Predicted band size: 21 kDa
Observed band size: 21 kDa
Western blot - Anti-Ras antibody [EPR23474-20] (ab275875)
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
Exposure time: 15 seconds.
All lanes: Western blot - Anti-Ras antibody [EPR23474-20] (ab275875) at 1/1000 dilution
Lane 1: His-tagged human KRAS recombinant protein, 20 ng
Lane 2: His-tagged human HRAS recombinant protein, 20 ng
Lane 3: His-tagged human NRAS recombinant protein, 20 ng
Secondary
All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Predicted band size: 21 kDa
Flow Cytometry (Intracellular) - Anti-Ras antibody [EPR23474-20] (ab275875)
Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling Ras with ab275875 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).
A Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.
Western blot - Anti-Ras antibody [EPR23474-20] (ab275875)
Blocking and diluting buffer and concentration: 5% NFDM/TBST
This antibody specifically recognizes RAS (mutated G12V).
In Western blot, anti-Ras antibody (ab275875) staining at 1/1000 dilution.
In Western blot, anti-His antibody (Anti-6X His tag® antibody [EPR20547] - ChIP Grade ab213204) staining at 1/5000 dilution.
In Western blot, anti-GAPDH antibody (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) loading control staining at 1/200000 dilution.
Exposure time: 180 seconds
All lanes: Western blot - Anti-RAS (mutated G12V) antibody [EPR26090-62] (Anti-RAS (mutated G12V) antibody [EPR26090-62] ab313698) at 1/1000 dilution
Lane 1: 293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a His-tag whole cell lysate at 20 µg
Lane 2: 293T cells transfected with a human KRAS (mutated G12V) expression vector containing a His-tag whole cell lysate at 20 µg
Lane 3: 293T cells transfected with a human KRAS (mutated G12R) expression vector containing a His-tag whole cell lysate at 20 µg
Lane 4: 293T cells transfected with a human KRAS (mutated G13D) expression vector containing a His-tag whole cell lysate at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Observed band size: 21 kDa
Exposure time: 180s
Western blot - Anti-Ras antibody [EPR23474-20] (ab275875)
Blocking and diluting buffer and concentration: 5% NFDM/TBST
This antibody specifically recognizes RAS (mutated G12V).
In Western blot, anti-Ras antibody (ab275875) staining at 1/1000 dilution.
In Western blot, anti-His antibody (Anti-6X His tag® antibody [EPR20547] - ChIP Grade ab213204) staining at 1/5000 dilution.
In Western blot, anti-GAPDH antibody (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) loading control staining at 1/200000 dilution.
Exposure time: 180 seconds
All lanes: Western blot - Anti-RAS (mutated G12V) antibody [EPR26090-62] (Anti-RAS (mutated G12V) antibody [EPR26090-62] ab313698) at 1/1000 dilution
Lane 1: 293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a His-tag whole cell lysate at 20 µg
Lane 2: 293T cells transfected with a human KRAS (mutated G12V) expression vector containing a His-tag whole cell lysate at 20 µg
Lane 3: 293T cells transfected with a human HRAS (mutated G12V) expression vector containing a His-tag whole cell lysate at 20 µg
Lane 4: 293T cells transfected with a human NRAS (mutated G12V) expression vector containing a His-tag whole cell lysate at 20 µg
Lane 5: 293T cells transfected with a human KRAS expression vector containing a His-tag whole cell lysate at 40 µg
Lane 6: 293T cells transfected with a human KRAS (mutated G12C) expression vector containing a His-tag whole cell lysate at 40 µg
Lane 7: 293T cells transfected with a human KRAS (mutated G12A) expression vector containing a His-tag whole cell lysate at 20 µg
Lane 8: 293T cells transfected with a human KRAS (mutated G12D) expression vector containing a His-tag whole cell lysate at 20 µg
Lane 9: 293T cells transfected with a human KRAS (mutated G12S) expression vector containing a His-tag whole cell lysate at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Observed band size: 21 kDa
Exposure time: 180s
Western blot - Anti-Ras antibody [EPR23474-20] (ab275875)
Blocking and diluting buffer and concentration: 5% NFDM/TBST
Positive control: SW620, SW480, CAPAN-1 (KRAS G12V)

Negative control: THP-1 (N-RAS G12D), HCT 116 (K-RAS G13D), Calu-1 (K-RAS G12C), A549 (K-RAS G12S), Caco-2 (K-RAS wild-type) and HT-29 (K-RAS wild-type).

This antibody specifically recognizes RAS (mutated G12V).

In Western blot, anti-Ras antibody (ab275875) staining at 1/1000 dilution.

In Western blot, anti-GAPDH antibody (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) loading control staining at 1/200000 dilution.
Exposure time: 180 seconds
All lanes: Western blot - Anti-RAS (mutated G12V) antibody [EPR26090-62] (Anti-RAS (mutated G12V) antibody [EPR26090-62] ab313698) at 1/1000 dilution
Lane 1: SW620 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 48 µg
Lane 2: SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 48 µg
Lane 3: Capan-1 (human pancreatic adenocarcinoma epithelial cell) whole cell lysate at 48 µg
Lane 4: THP-1 (human monocytic leukemia monocyte) whole cell lysate at 48 µg
Lane 5: HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 48 µg
Lane 6: Calu-1 (human lu epidermoid carcinoma epithelial cell) whole cell lysate 48 at 48 µg
Lane 7: A549 (human lung carcinoma epithelial cell) whole cell lysate 48 at 48 µg
Lane 8: Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 48 µg
Lane 9: HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 48 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Observed band size: 21 kDa
Exposure time: 180s
Western blot - Anti-Ras antibody [EPR23474-20] (ab275875)
Blocking and diluting buffer and concentration: 5% NFDM/TBST
Positive control: SW620, SW480, (KRAS G12V)

Negative control: SW1116 (KRAS G12A), RPMI-8226 (KRAS G12A), MIA PaCa-2 (KRAS G12C), LoVo (KRAS G13D).

This antibody specifically recognizes RAS (mutated G12V).

In Western blot, anti-Ras antibody (ab275875) staining at 1/1000 dilution.

In Western blot, anti-GAPDH antibody (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) loading control staining at 1/200000 dilution.
Exposure time: 180 seconds
All lanes: Western blot - Anti-RAS (mutated G12V) antibody [EPR26090-62] (Anti-RAS (mutated G12V) antibody [EPR26090-62] ab313698) at 1/1000 dilution
Lane 1: SW620 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 48 µg
Lane 2: SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 48 µg
Lane 3: SW1116 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 48 µg
Lane 4: RPMI-8226 (human plasmacytoma B Lymphocyte) whole cell lysate at 48 µg
Lane 5: MIA PaCa-2 (human pancreas epithelial cell) whole cell lysate at 48 µg
Lane 6: LoVo (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 48 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Observed band size: 21 kDa
Exposure time: 180s
Flow Cytometry (Intracellular) - Anti-Ras antibody [EPR23474-20] (ab275875)
Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling Ras with ab275875 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).
A Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.
Multiplex immunohistochemistry - Anti-Ras antibody [EPR23474-20] (ab275875)
Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse stomach tissue staining VAChT with Anti-VAChT antibody [EPR29154-71] ab317452 at 1/1000 dilution, ab322711 anti-ALDH3A1 used at 1/500 dilution and ab275875 anti-Ras used at 1/2000 dilution. Opal Polymer HRP Ms + Rb was used as a secondary antibody.
Panel A: merged staining of anti-VAChT (green; Opal^™520), anti-ALDH3A1 (gray; Opal^™570) and anti-Ras (magenta; Opal^™690) on mouse stomach.

Panel B: anti-VAChT staining nerve plexus in mouse stomach.

Panel C: ant-ALDH3A1 staining epithelium in mouse stomach.

Panel D: ant-Ras showed mainly membranous staining in mouse stomach.

Nuclear DNA was labeled with DAPI (shown in blue).
The section was incubated in three rounds of staining: in the order of Anti-VAChT antibody [EPR29154-71] ab317452, ab322711 and ab275875 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BOND^®RX instrument with an Opal^™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.