Anti-HMGB2 antibody [EPR6302]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGB2 antibody [EPR6302] (AB133540)

Immunohistochemistry analysis of paraffin embedded Human breast tissue labelling HMGB2 with ab133540 at 1/250 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGB2 antibody [EPR6302] (AB133540)

Immunohistochemistry analysis of paraffin embedded Human colon carcinoma tissue labelling HMGB2 with ab133540 at 1/250 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-HMGB2 antibody [EPR6302] (AB133540)

Lanes 1- 2 : Merged signal (red and green). Green - ab133540 observed at 24 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab133540 was shown to react with HMGB2 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266358 (knockout cell lysate ab257156) was used. Wild-type HEK-293T and HMGB2 knockout HEK-293T 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. ab133540 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.

All lanes:

Western blot - Anti-HMGB2 antibody [EPR6302] (ab133540) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

HMGB2 knockout HEK-293T cell lysate at 20 µg

Predicted band size: 24 kDa

Observed band size: 24 kDa

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• WB

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Western blot - Anti-HMGB2 antibody [EPR6302] (AB133540)

All lanes:

Western blot - Anti-HMGB2 antibody [EPR6302] (ab133540) at 1/1000 dilution

Lane 1:

K562 cell lysate at 10 µg

Lane 2:

HL-60 cell lysate at 10 µg

Lane 3:

C6 cell lysate at 10 µg

Lane 4:

RAW 264.7 cell lysate at 10 µg

Lane 5:

PC-12 cell lysate at 10 µg

Predicted band size: 24 kDa

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• WB

Lab

Western blot - Anti-HMGB2 antibody [EPR6302] (AB133540)

Lanes 1 - 4 : Merged signal (red and green). Green - ab133540 observed at 24 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab133540 was shown to recognize HMGB2 in wild-type HAP1 cells as signal was lost at the expected MW in HMGB2 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and HMGB2 knockout samples were subjected to SDS-PAGE. ab133540 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 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:

Western blot - Anti-HMGB2 antibody [EPR6302] (ab133540) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

HMGB2 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

K562 cell lysate at 20 µg

Predicted band size: 24 kDa

Observed band size: 24 kDa

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-HMGB2 antibody [EPR6302] (AB133540)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.