Anti-HLA A antibody [EP1395Y] - Low endotoxin, Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HLA A antibody [EP1395Y] - Low endotoxin, Azide free (AB246691)

This data was developed using the same antibody clone in a different buffer formulation (ab52922).

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling Anti-HLA A with ab52922 at a concentration of 0.01 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

Anti-HLA A antibody [EP1395Y] ab52922 was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-HLA A antibody [EP1395Y] - Low endotoxin, Azide free (AB246691)

ICC/IF image of unpurified ab52922 stained MCF7 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab52922, 5μg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HLA A antibody [EP1395Y] - Low endotoxin, Azide free (AB246691)

ab52922 at 1/250 dilution staining human tonsil; paraffin embedded.

• IHC-P

AbReview38970****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HLA A antibody [EP1395Y] - Low endotoxin, Azide free (AB246691)

ab52922 staining HLA A in Human tonsil tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 3% H₂O₂ for 10 minutes at 25°C; antigen retrieval was by heat mediation in a citrate buffer, pH 6.0 . Samples were incubated with primary antibody (1/3000) for 20 minutes at 25°C. An undiluted HRP-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).

This image is courtesy of an anonymous Abreview.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-HLA A antibody [EP1395Y] - Low endotoxin, Azide free (AB246691)

Immunocytochemistry/Immunofluorescence analysis of Raji (human Burkitt's lymphoma) cells labelling HLA A with purified ab52922 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1 : primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HLA A antibody [EP1395Y] - Low endotoxin, Azide free (AB246691)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling HLA A with purified ab52922 at 1/100. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, a goat anti-rabbit IgG H&L (HRP) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-HLA A antibody [EP1395Y] - Low endotoxin, Azide free (AB246691)

Intracellular Flow Cytometry analysis ofRaji cells labelling HLA Awith purified ab52922 at 1/40 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-HLA A antibody [EP1395Y] - Low endotoxin, Azide free (AB246691)

Overlay histogram showing Raji cells stained with ab52922 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab52922, 1/100) for 30 min at 22°C. The secondary antibody used was DyLight© 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Raji cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).

• IP

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Immunoprecipitation - Anti-HLA A antibody [EP1395Y] - Low endotoxin, Azide free (AB246691)

ab52922 (purified) at 1/20 immunoprecipitating HLA A in A549 whole cell lysate. 10 ug of cell lysate was present in the input. For western blotting, a HRP-conjugated Veriblot for IP Detection Reagent (ab131366) was used for detection at 1/10,000 dilution. A rabbit monoclonal IgG (ab172730) was used intead of ab128913 as a negative control (Lane 3).

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).

All lanes:

Immunoprecipitation - Anti-HLA A antibody [EP1395Y] (<a href='/en-us/products/primary-antibodies/hla-a-antibody-ep1395y-ab52922'>ab52922</a>)

Predicted band size: 41 kDa

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• IP

Unknown

Immunoprecipitation - Anti-HLA A antibody [EP1395Y] - Low endotoxin, Azide free (AB246691)

ab52922 (purified) at 1/20 immunoprecipitating HLA A in THP-1 whole cell lysate. 10 ug of cell lysate was present in the input. For western blotting, a HRP-conjugated Veriblot for IP Detection Reagent (ab131366) was used for detection at 1/10,000 dilution. A rabbit monoclonal IgG (ab172730) was used intead of ab128913 as a negative control (Lane 3).

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).

All lanes:

Immunoprecipitation - Anti-HLA A antibody [EP1395Y] (<a href='/en-us/products/primary-antibodies/hla-a-antibody-ep1395y-ab52922'>ab52922</a>)

Predicted band size: 41 kDa

false

• WB

Lab

Western blot - Anti-HLA A antibody [EP1395Y] - Low endotoxin, Azide free (AB246691)

This data was developed using the same antibody clone in a different buffer formulation (ab52922). Anti-HLA-A antibody [EP1395Y] (ab52922) staining at 1/10000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab52922 was shown to bind specifically to HLA-A. A band was observed at 41 kDa in wild-type A549 cell lysates with no signal observed at this size in HLA-A knockout cell line. To generate this image, wild-type and HLA-A knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-HLA A antibody [EP1395Y] (<a href='/en-us/products/primary-antibodies/hla-a-antibody-ep1395y-ab52922'>ab52922</a>) at 1/10000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Western blot - Human HLA-A knockout A549 cell line (<a href='/en-us/products/cell-lines/human-hla-a-knockout-a549-cell-line-ab287473'>ab287473</a>)

Lane 2:

HLA-A knockout A549 cell lysate at 20 µg

Lane 3:

Wild-type A431 cell lysate at 20 µg

Lane 4:

HLA-A knockout A431 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 41 kDa

Observed band size: 41 kDa

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• WB

Supplier Data

Western blot - Anti-HLA A antibody [EP1395Y] - Low endotoxin, Azide free (AB246691)

This data was developed using the same antibody clone in a different buffer formulation (ab52922).

Lanes 1 - 4 : Merged signal (red and green). Green - ab52922 observed at 40 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab52922 was shown to react with HLA-A in A431 wild-type cells in Western blot. Loss of signal was observed when HLA-A knockout sample was used. A431 wild-type and HLA-A knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween^®) before incubation with ab52922 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-HLA A antibody [EP1395Y] (<a href='/en-us/products/primary-antibodies/hla-a-antibody-ep1395y-ab52922'>ab52922</a>) at 1/10000 dilution

Lane 1:

Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

HLA A knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human HLA-A knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-hla-a-knockout-a-431-cell-line-ab261894'>ab261894</a>)

Lane 3:

A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

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