Anti-V5 tag antibody [EPR27187-61] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-V5 tag antibody [EPR27187-61] - BSA and Azide free (AB309486)

This data was developed using ab309485, the same antibody clone in a different buffer formulation. Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293T (human embryonic kidney epithelial cell) transfected with a CBX1-V5-tagged expression vector containing a myc tag cells labelling V5 tag with ab309485 at 1/500 dilution (0.1 ug)/Right (Red) compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-V5 tag antibody [EPR27187-61] - BSA and Azide free (AB309486)

This data was developed using ab309485, the same antibody clone in a different buffer formulation. Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEK-293T (human embryonic kidney epithelial cell) cells labelling V5 tag with ab309485 at 1/500 (0.992 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing positive staining in HEK-293T cells transfected a CBX1-V5-tagged expression vector containing a myc tag.Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. ab223894 Anti-Myc tag mouse monoclonal antibody (Alexa Fluor® 594) was used to counterstain tubulin 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 1/1000 2 ug/ml dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-V5 tag antibody [EPR27187-61] - BSA and Azide free (AB309486)

This data was developed using ab309485, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded (A) HEK-293T (human tissue labeling V5 tag with ab309485 at 1/2000 (0.248 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 His tag construct, no staining on (B) HEK-293T transfected with empty plasmid. The section was incubated with ab309485 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 Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-V5 tag antibody [EPR27187-61] - BSA and Azide free (AB309486)

This data was developed using ab309485, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling V5 tag with ab309485 at 1/2000 (0.248 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 ab309485 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 Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-V5 tag antibody [EPR27187-61] - BSA and Azide free (AB309486)

This data was developed using ab309485, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling V5 tag with ab309485 at 1/2000 (0.248 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 ab309485 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 Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-V5 tag antibody [EPR27187-61] - BSA and Azide free (AB309486)

This data was developed using ab309485, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling V5 tag with ab309485 at 1/2000 (0.248 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 ab309485 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 Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IP

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Immunoprecipitation - Anti-V5 tag antibody [EPR27187-61] - BSA and Azide free (AB309486)

This data was developed using ab309485, the same antibody clone in a different buffer formulation.

V5 tag was immunoprecipitated from 0.35 mg 293T cells transfected with a V5 tag-Chromobox protein homolog 1 expression vector containing a His-tag whole cell lysate with ab309485 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab309485 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : 293T cells transfected with a V5 tag-Chromobox protein homolog 1 expression vector containing a His-tag whole cell lysate
Lane 2 : ab309485 IP in 293T cells transfected with a V5 tag-Chromobox protein homolog 1 expression vector containing a His-tag whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab309485 in 293T cells transfected with a V5 tag-Chromobox protein homolog 1 expression vector containing a His-tag whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 seconds

All lanes:

Immunoprecipitation - Anti-V5 tag antibody [EPR27187-61] (<a href='/en-us/products/primary-antibodies/v5-tag-antibody-epr27187-61-ab309485'>ab309485</a>) at 1/1000 dilution

All lanes:

293T cells transfected with a V5 tag-Chromobox protein homolog 1 expression vector containing a His-tag whole cell lysate at 10 µg

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

• WB

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Western blot - Anti-V5 tag antibody [EPR27187-61] - BSA and Azide free (AB309486)

This data was developed using ab309485, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST In Western blot, anti-His antibody (ab213204) staining at 1/5000 dilution. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 81 seconds

All lanes:

Western blot - Anti-V5 tag antibody [EPR27187-61] (<a href='/en-us/products/primary-antibodies/v5-tag-antibody-epr27187-61-ab309485'>ab309485</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

293T cells transfected with a V5 tag-Chromobox protein homolog 1 expression vector containi a His-tag whole cell lysate at 10 µg

Lane 3:

His-tagged human Chromobox protein homolog 1 recombinant protein at 50 ng

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: 81s