Anti-HA tag antibody [RM1058]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-HA tag antibody [RM1058] (AB314237)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype (Left) / 293T cells transfected with a human HA Tag expression vector containing a GFP tag (Middle) / 293T cells transfected with an empty expression vector containing a GFP tag (Right) cells labelling HA tag with ab314237 at 1/5000 dilution (0.01 ug)/Middle and Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control.

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HA tag antibody [RM1058] (AB314237)

Immunohistochemical analysis of paraffin-embedded (A) HEK-293T (human embryonic kidney epithelial cell) transfected with a HA Tag expression vector. (B) HEK-293T transfected with empty vector. tissue labeling HA tag with ab314237 at 1/5000 (0.104 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) HEK-293T (human embryonic kidney epithelial cell) transfected with a HA Tag expression vector. No staining on (B) HEK-293T transfected with empty vector.
The section was incubated with ab314237 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-HA tag antibody [RM1058] (AB314237)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEK-293T (human epithelial cell line from embryonic kidney) transfected with GFP-tagged human HA Tag expression vector cells labelling HA tag with ab314237 at 1/500 (1.042 ug/ml) dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) antibody at 1/1000 (2 ug/mL) dilution (Green).

Confocal image showing cytoplasmic staining in 293T cells transfected with a human HA Tag expression vector containing a GFP tag.
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/mL) dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HA tag antibody [RM1058] (AB314237)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling HA tag with ab314237 at 1/5000 (0.104 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on human tonsil.
The section was incubated with ab314237 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.

• ChIP

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ChIP - Anti-HA tag antibody [RM1058] (AB314237)

Chromatin was prepared from Hela (human epithelial cell line from cervix adenocarcinoma) transfected with HA Tagged human CREBBP and Hela transfected with empty vector cells according to the Abcam Dual-X-ChIP protocol*.Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.

The ChIP was performed with 25 µg of chromatin, 5 µg of ab314237 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (SYBR green approach).

*http : //www.abcam.com/resources?keywords=X%20ChIP%20protocol

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HA tag antibody [RM1058] (AB314237)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling HA tag with ab314237 at 1/5000 (0.104 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on mouse spleen.
The section was incubated with ab314237 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-HA tag antibody [RM1058] (AB314237)

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling HA tag with ab314237 at 1/5000 (0.104 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on rat spleen.
The section was incubated with ab314237 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-HA tag antibody [RM1058] (AB314237)

HA tag was immunoprecipitated from 0.35 mg 293T (human embryonic kidney) transfected with TIM 4 (WT) expression vector containing a HA-myc-His-GFP-tag and PDGFR TM domain, whole cell lysate with ab314237 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314237 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : 293T (human embryonic kidney) transfected with TIM 4 (WT) expression vector containing a HA-myc-His-GFP-tag and PDGFR TM domain, whole cell lysate
Lane 2 : ab314237 IP in 293T (human embryonic kidney) transfected with TIM 4 (WT) expression vector containing a HA-myc-His-GFP-tag and PDGFR TM domain, whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab314237 in 293T transfected with TIM 4 (WT) expression vector containing a HA-myc-His-GFP-tag and PDGFR TM domain, whole cell lysate

All lanes:

Immunoprecipitation - Anti-HA tag antibody [RM1058] (ab314237) at 1/30 dilution

All lanes:

293T (human embryonic kidney) transfected with TIM 4 (WT) expression vector containing a HA-myc-His-GFP-tag and PDGFR TM domain, 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: 8s

• WB

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Western blot - Anti-HA tag antibody [RM1058] (AB314237)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-GFP antibody [E385] (ab32146) staining at 1/10000 dilution.

In Western blot, Anti-TIM 4 antibody [EPR22304-3] (ab222093) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-HA tag antibody [RM1058] (ab314237) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney) transfected with an empty vector (vector control), containing a myc-GFP-tag, whole cell lysate at 20 µg

Lane 2:

293T transfected with TIM 4 (WT) expression vector containing a HA-myc-His-GFP-tag and PDGFR TM domain, 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 1/100000 dilution

Observed band size: 78 kDa,36 kDa

false

Exposure time: 37s