Anti-HMGB1/Hmg 1 Rabbit Monoclonal Antibody

Figure 1. Western blot analysis of HMGB1 using anti-HMGB1 antibody (M00066-1).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human Hela whole cell lysates,
Lane 2: human 293T whole cell lysates,
Lane 3: human K562 whole cell lysates,
Lane 4: human Jurkat whole cell lysates,
Lane 5: rat brain tissue lysates,
Lane 6: rat liver tissue lysates,
Lane 7: mouse brain tissue lysates,
Lane 8: mouse liver tissue lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-HMGB1 antigen affinity purified monoclonal antibody (Catalog # M00066-1) at 1:1000 overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:1000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for HMGB1 at approximately 25 kDa. The expected band size for HMGB1 is at 25 kDa.

Figure 2. IHC analysis of HMGB1 using anti-HMGB1 antibody (M00066-1).
HMGB1 was detected in a paraffin-embedded section of human colorectal adenocarcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:50 rabbit anti-HMGB1 Antibody (M00066-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Figure 3. IHC analysis of HMGB1 using anti-HMGB1 antibody (M00066-1).
HMGB1 was detected in a paraffin-embedded section of human lung adenocarcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:50 rabbit anti-HMGB1 Antibody (M00066-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Figure 4. IHC analysis of HMGB1 using anti-HMGB1 antibody (M00066-1).
HMGB1 was detected in a paraffin-embedded section of human spleen tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:50 rabbit anti-HMGB1 Antibody (M00066-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Figure 5. IHC analysis of HMGB1 using anti-HMGB1 antibody (M00066-1).
HMGB1 was detected in a paraffin-embedded section of human prostatic acinar adenocarcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:50 rabbit anti-HMGB1 Antibody (M00066-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Figure 6. IHC analysis of HMGB1 using anti-HMGB1 antibody (M00066-1).
HMGB1 was detected in a paraffin-embedded section of human cervical squamous carcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:50 rabbit anti-HMGB1 Antibody (M00066-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Figure 7. IF analysis of HMGB1 using anti-HMGB1 antibody (M00066-1) and anti-Beta Tubulin antibody (M01857-3).
HMGB1 was detected in immunocytochemical section of Hela cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated at 1:50 with rabbit anti-HMGB1 Antibody (M00066-1) and mouse anti-Beta Tubulin antibody (M01857-3) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) and Cy3 Conjugated Goat Anti-Mouse IgG (BA1031) were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Immunohistochemical analysis of paraffin-embedded Rat liver, using the Antibody at 1:300 dilution.

Immunohistochemical analysis of paraffin-embedded Rat kidney, using the Antibody at 1:300 dilution.

Immunohistochemical analysis of paraffin-embedded Mouse skeletal muscle - gastrocnemius , using the Antibody at 1:300 dilution.