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?

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.

ab79823 staining HMGB1 in wild-type HAP1 cells (top panel) and HMGB1 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour. The cells were then incubated with ab79823 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1 hour with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labeling 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.

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

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.

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.

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.

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).

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

Lane 1 : SK-BR-3 (Human mammary gland adenocarcinoma cell line) cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 25 kDa
Observed band size: 25 kDa

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

Anti-HMGB1 antibody [EPR3507] (ab79823) at 1/10000 dilution (purified) + Rat brain tissue lysate at 10 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 25 kDa
Observed band size: 25 kDa

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

All lanes : Anti-HMGB1 antibody [EPR3507] (ab79823) at 1/50000 dilution (unpurified)

Lane 1 : SK-BR-3 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : HepG2 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-rabbit HRP conjugate at 1/2000 dilution

Predicted band size: 25 kDa
Observed band size: 25 kDa

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

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

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.