- MA5-17277 - Invitrogen Antibodies HMGB1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of HMGB1 using A) 30 µg Neuro2A whole cell lysate (B) 30 µg C8D30 whole cell lysate (C) 30 µg NIH-3T3 whole cell lysate (D) 30 µg BCL-1 whole cell lysate (E) 30 µg Raw264.7 whole cell lysate and F) 30 µg C2C12 whole cell lysate. Samples were loaded onto a 12% SDS-PAGE gel and probed with a HMGB1 monoclonal antibody (Product # MA5-17277) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot analysis of HMGB1 was performed by separating 30 µg of non-transfected (–) and transfected (+) 293T whole cell extracts by 12% SDS-PAGE. Proteins were transferred to a membrane and probed with a HMGB1 Monoclonal Antibody (GT383) (Product # MA5-17277) at a dilution of 1:3000. The HRP-conjugated anti-mouset IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: HMGB1 Monoclonal Antibody (GT383) detects HMGB1 protein by western blot analysis. A. 30 µg 293T whole cell lysate/extract. B. 30 µg A431 whole cell lysate/extract. C. 30 µg HeLa whole cell lysate/extract. D. 30 µg HepG2 whole cell lysate/extract. E. 30 µg A375 whole cell lysate/extract.12% SDS-PAGE. HMGB1 Monoclonal Antibody (GT383) (Product # MA5-17277) dilution: 1:1,000. The HRP-conjugated anti-mouse IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of HMGB1 was performed by separating 50 µg of various tissue extracts by 12% SDS-PAGE. Proteins were transferred to a membrane and probed with a HMGB1 Monoclonal Antibody (GT383) (Product # MA5-17277) at a dilution of 1:2000. The HRP-conjugated anti-mouse IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: HMGB1 Monoclonal Antibody (GT383) detects HMGB1 protein by western blot analysis. A. 30 µg PC-12 whole cell lysate/extract. B. 30 µg Rat-2 whole cell lysate/extract.12% SDS-PAGE. HMGB1 Monoclonal Antibody (GT383) (Product # MA5-17277) dilution: 1:1,000. The HRP-conjugated anti-mouse IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of HMGB1 was performed using 90% confluent log phase SH-SY5Y cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with HMGB1 Monoclonal Antibody (GT383) (Product # MA5-17277) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry-Immunofluorescence analysis of HMGB1 was performed in DIV9 rat E18 primary cortical neurons fixed in 4% paraformaldehyde at RT for 15 min. Green: HMGB1 Monoclonal Antibody (GT383) (Product # MA5-17277) diluted at 1:500. Red: MAP2, stained by MAP2 antibody. Blue: Fluoroshield with DAPI.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of HMGB1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR784665_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Immunofluorescence analysis was performed on wild type HeLa cells (panel a,d), HeLa Cas9 control cells (panel b,e) and HeLa HMGB1KO cells (panel c, f). Cells were fixed, permeabilized, and labeled with HMGB1 Monoclonal Antibody (GT383) (Product # MA5-17277, 1:150 dilution), followed by Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (Product # A32723, 1:2000). Nuclei (blue) were stained using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Loss of signal (panel c,f) upon CRISPR mediated knockout (KO) confirms that antibody is specific to HMGB1 (green). The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry (Frozen) analysis of HMGB1 was performed in frozen-sectioned adult mouse cerebellum tissue using HMGB1 Monoclonal Antibody (GT383) (Product # MA5-17277) at a dilution of 1:250 (Green). Red: Calbindin, stained by Calbindin antibody diluted at 1:500. Blue: Fluoroshield with DAPI. Antigen Retrieval: Citrate buffer, pH 6.0, 5 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: HMGB1 Monoclonal Antibody (GT383) detects HMGB1 protein at nucleus on mouse brain by immunohistochemical analysis. Sample: Paraffin-embedded mouse brain. HMGB1 Monoclonal Antibody (GT383) (Product # MA5-17277) dilution: 1:200. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: HMGB1 Monoclonal Antibody (GT383) detects HMGB1 protein at nucleus on SAS xenograft by immunohistochemical analysis. Sample: Paraffin-embedded SAS xenograft. HMGB1 Monoclonal Antibody (GT383) (Product # MA5-17277) dilution: 1:200. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

MA5-17277