All lanes : Anti-HMGB1 antibody (ab18256) at 1 µg/ml

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : HMGB1 knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 25 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab18256 observed at 29 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab18256 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. Ab18256 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml 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.

ab18256 stained in HeLa cells. Cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab18256 at 1µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) (pseudo-colored red) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature

All lanes : Anti-HMGB1 antibody (ab18256) at 1 µg/ml

Lane 1 : NIH/3T3 whole cell lysate (ab7179)
Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 3 : PC-12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lanes 1 & 3 : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution
Lane 2 : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 25 kDa
Observed band size: 29 kDa

why is the actual band size different from the predicted?
Additional bands at: 59 kDa. We are unsure as to the identity of these extra bands.

All lanes : Anti-HMGB1 antibody (ab18256) at 1 µg/ml

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : Jurkat whole cell lysate (ab7899)
Lane 3 : A-431 whole cell lysate (ab7909)
Lane 4 : HEK-293 whole cell lysate (ab7902)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 25 kDa
Observed band size: 29 kDa why is the actual band size different from the predicted?

Anti-HMGB1 antibody (ab18256) at 1 µg/ml + Recombinant Human HMGB1 protein (ab56525) at 0.01 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 25 kDa


Exposure time: 2 minutes

All lanes : Anti-HMGB1 antibody (ab18256) at 1/1000 dilution

Lane 1 : Rat brain whole tissue lysate - infused with asf for 1 week
Lane 2 : Rat brain whole tissue lysate - infused with LPS for 1 week
Lane 3 : Rat brain whole tissue lysate - infused with acsf for 8 weeks
Lane 4 : Rat brain whole tissue lysate - infused with LPS for 8 weeks
Lane 5 : Rat brain whole tissue lysate - infused with LPS for 4 weeks
Lane 6 : Rat brain whole tissue lysate -infused with LPS for 4 weeks, after 2 weeks of LPS infusion were treated with neramexane for the next 2 weeks
Lane 7 : Rat brain whole tissue lysate - infused with LPS for 4 weeks, after 2 weeks of LPS infusion were treated with memantine for the next 2 weeks.

Lysates/proteins at 40 µg per lane.

Secondary
All lanes : Biotinylated Goat anti-rabbit IgG

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 25 kDa


Exposure time: 30 seconds

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