Anti-HMGB1 antibody [EPR3507] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-HMGB1 antibody [EPR3507] - BSA and Azide free (AB216986)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab79823).

Flow cytometry overlay histogram showing wild-type Hap1 (green line) and HMGB1 knockout Hap1 stained with ab79823 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab79823) (1x 106 in 100μl at 0.008 μg/ml (1/260000)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type Hap1 - black line, HMGB1 knockout Hap1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-HMGB1 antibody [EPR3507] - BSA and Azide free (AB216986)

Overlay histogram showing HeLa (Human epithelial cell line from cervix adenocarcinoma) cells stained with unpurified ab79823 (red line). The cells were fixed with 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 (ab79823, 1/20 dilution) 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 monoclonal IgG (0.5μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in HeLa cells fixed with 4% paraformaldehyde/permeabilized with 0.1% PBS-Tween 20 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 (ab79823).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGB1 antibody [EPR3507] - BSA and Azide free (AB216986)

This IHC data was generated using the same anti-HMGB1 antibody clone, EPR3507, in a different buffer formulation (cat# ab79823).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling HMGB1 with unpurified ab79823 at 1/350. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with Hematoxylin.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-HMGB1 antibody [EPR3507] - BSA and Azide free (AB216986)

Clone EPR3507 (ab216986) has been successfully conjugated by Abcam. This image was generated using Anti-HMGB1 antibody [EPR3507] (PE). Please refer to ab225042 for protocol details.

Overlay histogram showing HeLa cells stained with ab225042 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab225042, 1/1000 dilution) for 30 min at 22°C.

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGB1 antibody [EPR3507] - BSA and Azide free (AB216986)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labeling HMGB1 with unpurified ab79823 at 1/250 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab79823).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] - BSA and Azide free (AB216986)

Clone EPR3507 (ab216986) has been successfully conjugated by Abcam. This image was generated using Anti-HMGB1 antibody [EPR3507] (Alexa Fluor® 647). Please refer to ab195011 for protocol details.

ab195011 staining HMGB1 in HeLa cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab195011 at 1/100 dilution(shown in red) and ab195887, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor® 488, shown in green) at 2µg/ml overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

This product gave a positive signal in 100% methanol (5 min) fixed HeLa cells under the same testing conditions.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] - BSA and Azide free (AB216986)

Clone EPR3507 (ab216986) has been successfully conjugated by Abcam. This image was generated using Anti-HMGB1 antibody [EPR3507] (Alexa Fluor® 488). Please refer to ab195010 for protocol details.

ab195010 staining HMGB1 in HeLa cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab195010 at a working dilution of 1/100 (shown in green) and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor® 594, shown in red) at 2 µg/ml overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

This product gave a positive signal in 100% methanol (5 min) fixed HeLa cells under the same testing conditions.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] - BSA and Azide free (AB216986)

Immunocytochemsitry/Immunofluorescence analysis of HeLa cells labelling HMGB1 (red) with unpurified ab79823 at 1/350. Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 555-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. Counterstained with DAPI (blue).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab79823).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGB1 antibody [EPR3507] - BSA and Azide free (AB216986)

This data was developed using ab79823, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded mouse muscle tissue labeling HMGB1 with ab79823 at 1/350 dilution.

Positive staining on mouse muscle.

The section was incubated with ab79823 incubated at +4 °C overnight followed by a Goat Anti-Rabbit IgG H&L (HRP polymer) ready to use secondary. Counterstained with Hematoxylin.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGB1 antibody [EPR3507] - BSA and Azide free (AB216986)

This data was developed using ab79823, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded rat brain tissue labeling HMGB1 with ab79823 at 1/350 dilution.

Positive staining on rat brain.

The section was incubated with ab79823 incubated at +4 °C overnight followed by a Goat Anti-Rabbit IgG H&L (HRP polymer) ready to use secondary. Counterstained with Hematoxylin.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)

• WB

Lab

Western blot - Anti-HMGB1 antibody [EPR3507] - BSA and Azide free (AB216986)

This data was developed using the same antibody clone in a different buffer formulation (ab79823).

Lanes 1- 2 : Merged signal (red and green). Green - ab79823 observed at 30 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab79823 was shown to react with HMGB1 in wild-type HeLa cells in western blot. Loss of signal at the expected size was observed when CRISPR/Cas9 edited cell line ab255395 (CRISPR/Cas9 edited cell lysate ab263782) was used. The band observed in lane 2 below 25kDa may represent truncated forms and cleaved fragments. Wild-type HeLa and HMGB1 CRISPR/Cas9 edited HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab79823 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-HMGB1 antibody [EPR3507] (<a href='/en-us/products/primary-antibodies/hmgb1-antibody-epr3507-ab79823'>ab79823</a>) at 1/10000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

HMGB1 CRISPR/Cas9 edited HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human HMGB1 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-hmgb1-knockout-hela-cell-line-ab255395'>ab255395</a>)

Predicted band size: 24 kDa

Observed band size: 30 kDa

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• WB

Lab

Western blot - Anti-HMGB1 antibody [EPR3507] - BSA and Azide free (AB216986)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab79823).

Lanes 1 - 4 : Merged signal (red and green). Green - ab79823 observed at 30 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab79823 was shown to specifically react with HMGB1 in wild-type HAP1 cells as signal was lost in HMGB1 knockout cells. Wild-type and HMGB1 knockout samples were subjected to SDS-PAGE. ab79823 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-HMGB1 antibody [EPR3507] (<a href='/en-us/products/primary-antibodies/hmgb1-antibody-epr3507-ab79823'>ab79823</a>) at 1/10000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

HMGB1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

Jurkat whole cell lysate at 20 µg

Lane 4:

HeLa whole cell lysate at 20 µg

Predicted band size: 24 kDa

false

• WB

Lab

Western blot - Anti-HMGB1 antibody [EPR3507] - BSA and Azide free (AB216986)

This data was developed using ab79823, the same antibody clone in a different buffer formulation.

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602).

All lanes:

Western blot - Anti-HMGB1 antibody [EPR3507] (<a href='/en-us/products/primary-antibodies/hmgb1-antibody-epr3507-ab79823'>ab79823</a>) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse kidney tissue lysate at 20 µg

Lane 3:

Rat brain tissue lysate at 20 µg

Lane 4:

Rat kidney tissue 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 1/20000 dilution

Observed band size: 25 kDa,36 kDa

false

Exposure time: 3s