Anti-HLA E antibody [MEM-E/02]

• WB

Lab

Western blot - Anti-HLA E antibody [MEM-E/02] (AB2216)

Lanes 1-4 : Merged signal (red and green). Green - ab2216 observed at 40 kDa. Red - loading control ab181602 observed at 36 kDa.

ab2216 Anti-HLA E antibody [MEM-E/02] was shown to specifically react with HLA E in wild-type A549 cells. Loss of signal was observed when knockout cell line ab267080 (knockout cell lysate ab258452) was used. Wild-type and HLA E knockout samples were subjected to SDS-PAGE. ab2216 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated at room temperature for 2.5 hours at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-HLA E antibody [MEM-E/02] (ab2216) at 1/500 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

HLA-E knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human HLA-E (HLA E) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-hla-e-hla-e-knockout-a549-cell-line-ab267080'>ab267080</a>)

Lane 3:

THP-1 cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Predicted band size: 40 kDa

Observed band size: 40 kDa

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• Flow Cyt

Unknown

Flow Cytometry - Anti-HLA E antibody [MEM-E/02] (AB2216)

Overlay histogram showing HL60 cells stained with ab2216 (red line). The cells were fixed with 80% methanol (5 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 (ab2216, 1 μg/1x10⁶ cells for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was Mouse IgG1 [ICIGG1] (ab91353, 2 μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HLA E antibody [MEM-E/02] (AB2216)

IHC image of ab2216 staining in human tonsil formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab2216, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• WB

Lab

Western blot - Anti-HLA E antibody [MEM-E/02] (AB2216)

Lanes 1-3 : Merged signal (red and green). Green - ab2216 observed at 40 kDa. Red - loading control ab52901 observed at kDa.

ab2216 Anti-HLA E antibody [MEM-E/02] was shown to specifically react with HLA E in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267231 (knockout cell lysate ab258454) was used. Wild-type and HLA E knockout samples were subjected to SDS-PAGE. ab2216 and Anti-beta Tubulin [EP1331Y] - Microtubule Marker (ab52901) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-HLA E antibody [MEM-E/02] (ab2216) at 1/500 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

HLA-E knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human HLA-E (HLA E) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-hla-e-hla-e-knockout-hek-293t-cell-line-ab267231'>ab267231</a>)

Lane 3:

THP-1 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Predicted band size: 40 kDa

Observed band size: 40 kDa

false

• WB

Lab

Western blot - Anti-HLA E antibody [MEM-E/02] (AB2216)

Lanes 1-4 : Merged signal (red and green). Green - ab2216 observed at 40 kDa. Red - loading control ab181602 observed at 36 kDa.

ab2216 Anti-HLA E antibody [MEM-E/02] was shown to specifically react with HLA E in wild-type A549 cells. Loss of signal was observed when knockout cell line ab267081 (knockout cell lysate ab258453) was used. Wild-type and HLA E knockout samples were subjected to SDS-PAGE. ab2216 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated at room temperature for 2.5 hours at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-HLA E antibody [MEM-E/02] (ab2216) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

HLA-E knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human HLA-E (HLA E) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-hla-e-hla-e-knockout-a549-cell-line-ab267081'>ab267081</a>)

Lane 3:

THP-1 cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Predicted band size: 40 kDa

Observed band size: 40 kDa

false