Anti-HA tag antibody [RM1058] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-HA tag antibody [RM1058] - BSA and Azide free (AB314238)

This data was developed using ab314237, the same antibody clone in a different buffer formulation.

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] - BSA and Azide free (AB314238)

This data was developed using ab314237, the same antibody clone in a different buffer formulation.

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] - BSA and Azide free (AB314238)

This data was developed using ab314237, the same antibody clone in a different buffer formulation.

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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HA tag antibody [RM1058] - BSA and Azide free (AB314238)

This data was developed using ab314237, the same antibody clone in a different buffer formulation.

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

Supplier Data

ChIP - Anti-HA tag antibody [RM1058] - BSA and Azide free (AB314238)

This data was developed using ab314237, the same antibody clone in a different buffer formulation.

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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HA tag antibody [RM1058] - BSA and Azide free (AB314238)

This data was developed using ab314237, the same antibody clone in a different buffer formulation.

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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HA tag antibody [RM1058] - BSA and Azide free (AB314238)

This data was developed using ab314237, the same antibody clone in a different buffer formulation.

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

Supplier Data

Immunoprecipitation - Anti-HA tag antibody [RM1058] - BSA and Azide free (AB314238)

This data was developed using ab314237, the same antibody clone in a different buffer formulation.

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] (<a href='/en-us/products/primary-antibodies/ha-tag-antibody-rm1058-ab314237'>ab314237</a>) 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

Supplier Data

Western blot - Anti-HA tag antibody [RM1058] - BSA and Azide free (AB314238)

This data was developed using ab314237, the same antibody clone in a different buffer formulation.

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] (<a href='/en-us/products/primary-antibodies/ha-tag-antibody-rm1058-ab314237'>ab314237</a>) 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