All lanes : Anti-HMGB1 antibody [EPR3506] (ab92310) at 1/1000 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 - ab92310 observed at 30 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab92310 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. ab92310 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 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.

ab92310 staining HMGB1 in the human cell line HeLa (human cervix adenocarcinoma) by flow cytometry. Cells were fixed with 4% paraformaldehyde, permeabilized with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/20. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

All lanes : Anti-HMGB1 antibody [EPR3506] (ab92310) at 1/1000 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 - ab92310 observed at 30 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab92310 was shown to recognize HMGB1 in wild-type HAP1 cells as signal was lost at the expected MW in HMGB1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and HMGB1 knockout samples were subjected to SDS-PAGE. ab92310 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 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 : Anti-HMGB1 antibody [EPR3506] (ab92310) at 1/1000 dilution

Lane 1 : SKBR3 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : Molt4 cell lysate

Lysates/proteins at 10 µg per lane.

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

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

ab92310 at 1/100 dilution staining HMGB1 in Human Liver by Immunohistochemistry, Paraffin-embedded tissue. The use of an HRP/AP polymerized antibody is recommended for a secondary antibody.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.