Anti-HMGB1 antibody [EPR3507]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] (AB79823)

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).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-HMGB1 antibody [EPR3507] (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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-HMGB1 antibody [EPR3507] (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).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-HMGB1 antibody [EPR3507] (AB79823)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGB1 antibody [EPR3507] (AB79823)

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.

• IHC

Lab

Immunohistochemistry - Anti-HMGB1 antibody [EPR3507] (AB79823)

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling HMGB1 with ab79823 at a concentration of 0.1µ/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab79823 anti-HMGB1 antibody [EPR3507] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• IHC

Lab

Immunohistochemistry - Anti-HMGB1 antibody [EPR3507] (AB79823)

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling HMGB1 with ab79823 at a concentration of 0.1µ/ml. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20mins.

ab79823 anti-HMGB1 antibody [EPR3507] was incubated for 15mins at room temperature. Sections were counterstained with Hematoxylin. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGB1 antibody [EPR3507] (AB79823)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGB1 antibody [EPR3507] (AB79823)

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)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGB1 antibody [EPR3507] (AB79823)

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)

• WB

Lab

Western blot - Anti-HMGB1 antibody [EPR3507] (AB79823)

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

All lanes:

Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823) at 1/10000 dilution

Lane 1:

SK-BR-3 (Human mammary gland adenocarcinoma cell line) cell lysate at 10 µg

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 24 kDa

Observed band size: 25 kDa

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• WB

Lab

Western blot - Anti-HMGB1 antibody [EPR3507] (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.

All lanes:

Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823) at 1/10000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

HMGB1 knockout HeLa cell lysate at 20 µg

Predicted band size: 24 kDa

Observed band size: 30 kDa

false

• WB

Lab

Western blot - Anti-HMGB1 antibody [EPR3507] (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] (ab79823) 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

false

• WB

Lab

Western blot - Anti-HMGB1 antibody [EPR3507] (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] (ab79823) 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] (AB79823)

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] (ab79823) 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

• WB

Lab

Western blot - Anti-HMGB1 antibody [EPR3507] (AB79823)

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

All lanes:

Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823) at 1/10000 dilution

All lanes:

Rat brain tissue lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 24 kDa

Observed band size: 25 kDa

false

• WB

Unknown

Western blot - Anti-HMGB1 antibody [EPR3507] (AB79823)

All lanes:

Western blot - Anti-HMGB1 antibody [EPR3507] (ab79823) at 1/50000 dilution

Lane 1:

SK-BR-3 cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

HepG2 cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP conjugate at 1/2000 dilution

Predicted band size: 24 kDa

Observed band size: 25 kDa

false