Anti-V5 tag antibody [RM1083]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-V5 tag antibody [RM1083] (AB314555)

Immunohistochemical analysis of paraffin-embedded (A) HEK-293T (human epithelial cell line from embryonic kidney) transfected with a CBX1-V5-tagged expression vector containing a His-tag. (B) HEK-293T transfected with empty vector containing a His-tag. Tissue labeling V5 tag with ab314555 at 1/20000 (0.027 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on (A) HEK-293T transfected with a CBX1-V5-tagged expression vector containing a His-tag. No staining on (B) HEK-293T transfected with empty vector containing a His-tag. The section was incubated with ab314555 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 LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-V5 tag antibody [RM1083] (AB314555)

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling V5 tag with ab314555 at 1/20000 (0.027 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on human liver. The section was incubated with ab314555 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 LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-V5 tag antibody [RM1083] (AB314555)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEK-293T (human epithelial cell line from embryonic kidney) transfected with a CBX1-V5-tagged expression vector containing a His-tag. cells labelling V5 tag with ab314555 at 1/500 (1.08 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1015 (2ug/ml) dilution (Green). Confocal image showing nuclear staining in 293T cells transfected with V5 tag protein expression vector containing a myc tag, no staining on HEK-293T transfected with empty vector containing a His-tag. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab223895 Anti-Myc tag Mouse monoclonal antibody (Alexa Fluor® 647) was used to counterstain at 1/100 (5ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1015 (2ug/ml) dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-V5 tag antibody [RM1083] (AB314555)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype (Left) / 293T cells transfected with V5 tag protein expression vector containing a myc tag (Middle) / 293T cells transfected with empty protein expression vector containing a myc tag (Right) cells labelling V5 tag with ab314555 at 1/500 dilution (0.1 ug)/Middle and Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody. Cells are co-stained with Myc tag conjugated to Alexa Fluor®647.

• IP

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Immunoprecipitation - Anti-V5 tag antibody [RM1083] (AB314555)

V5 tag was immunoprecipitated from 0.35 mg 293T cells transfected with a human CBX expression vector containing a myc-His-V5-tag, whole cell lysate with ab314555 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314555 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : 293T cells transfected with a human CBX expression vector containing a myc-His-V5-tag, whole cell lysate Lane 2 : ab314555 IP in 293T cells transfected with a human CBX expression vector containing a myc-His-V5-tag, whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab314555 in 293T cells transfected with a human CBX expression vector containing a myc-His-V5-tag, whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-V5 tag antibody [RM1083] (ab314555) at 1/30 dilution

All lanes:

293T cells transfected with a human CBX expression vector containing a myc-His-V5-tag, whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 3s

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-V5 tag antibody [RM1083] (AB314555)

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling V5 tag with ab314555 at 1/20000 (0.027 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on mouse liver. The section was incubated with ab314555 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 LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-V5 tag antibody [RM1083] (AB314555)

Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling V5 tag with ab314555 at 1/20000 (0.027 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on rat liver. The section was incubated with ab314555 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 LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

• WB

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Western blot - Anti-V5 tag antibody [RM1083] (AB314555)

Blocking and diluting buffer and concentration : 5% NFDM/TBST In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution. In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-V5 tag antibody [RM1083] (ab314555) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containi a myc-His-tag, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human CBX expression vector containi a myc-His-V5-tag, whole cell lysate at 20 µg

Lane 3:

His-tagged human Chromobox protein homolog 1 recombinant protein at 0.05 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

Observed band size: 27 kDa

false

Exposure time: 6s