All lanes : Anti-HMGB1 antibody [EPR3507] (ab79823) at 1/10000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : HMGB1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 25 kDa
Observed band size: 30 kDa why is the actual band size different from the predicted?

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 knockout cell line ab255395 (knockout 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 knockout 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.

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.

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).

All lanes : Anti-HMGB1 antibody [EPR3507] (ab79823) at 1/10000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : HMGB1 knockout HAP1 whole cell lysate
Lane 3 : Jurkat whole cell lysate
Lane 4 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 25 kDa

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.

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.

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).

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.

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).

Immunofluorescence analysis of murine RAW 264.7 macrophages, either untreated (upper panel) or treated with LPS (bottom panel). HMGB1 was stained using unpurified ab79823.
Cells were fixed in paraformaldehyde, blocked in BSA for 1h, followed by permeabilization in 10% Triton X-100 for 30 min. Samples were incubated with primary antibody overnight at 4°C. An Alexa Fluor^® 488-conjugated anti-rabbit IgG 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 (ab79823).

ICC/IF image of unpurified ab79823 stained DU 145 (Human prostate carcinoma cell line) 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 permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab79823 at 1/1000 dilution overnight at +4°C. The secondary antibody (green) was DyLight^® 488 goat anti- rabbit (ab96899) 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 (ab79823).

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).