ET1601-2

HMGB1 Recombinant Rabbit Monoclonal Antibody [SA39-03]

cat.: ET1601-2

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IF-Cell, IF-Tissue, IHC-P, FC
Clonality:	Monoclonal
Clone number:	SA39-03

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer:	1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration:	1ug/ul
Purification:	Protein A affinity purified.
Molecular weight:	Predicted band size: 25 kDa
Isotype:	IgG
Immunogen:	Synthetic peptide within Human HMGB1 aa 151-200 / 215.
Positive control:	HepG2 cell lysate, HeLa cell lysate, HCT 116 cell lysate, A549 cell lysate, Jurkat cell lysate, C2C12 cell lysate, C6 cell lysate, HeLa, mouse thymus tissue lysate, rat spleen tissue lysate, MCF-7, human tonsil tissue, human kidney tissue, mouse brain tissue, HCT 116, mouse hippocampus tissue.
Subcellular location:	Cytoplasm, Nucleus, Cell membrane, Secreted, Chromosome
Recommended Dilutions: WB IF-Cell IF-Tissue IHC-P FC	1:20,000-1:50,000 1:500 1:100 1:5,000-1:20,000 1:1,000
Uniprot #:	SwissProt: P09429 Human \| P63158 Mouse \| P63159 Rat
Alternative names:	Amphoterin Chromosomal protein, nonhistone, HMG1 DKFZp686A04236 High mobility group 1 High mobility group box 1 High mobility group protein 1 High mobility group protein B1 high-mobility group (nonhistone chromosomal) protein 1 HMG-1 HMG1 HMG3 HMGB 1 HMGB1 HMGB1_HUMAN NONHISTONE CHROMOSOMAL PROTEIN HMG1 SBP 1 Sulfoglucuronyl carbohydrate binding protein

Images

Fig1: Immunocytochemistry analysis of HeLa cells labeling HMGB1 with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/500 dilution and competitor's antibody at 1/250 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/500 dilution and competitor's antibody at 1/250 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Fig2: Western blot analysis of HMGB1 on different lysates with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/50,000 dilution and competitor's antibody at 1/10,000 dilution.

Lane 1: HepG2 cell lysate
Lane 2: HeLa cell lysate
Lane 3: HCT 116 cell lysate
Lane 4: A549 cell lysate
Lane 5: Jurkat cell lysate
Lane 6: C2C12 cell lysate
Lane 7: C6 cell lysate

Lysates/proteins at 15 µg/Lane.

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

Exposure time: 21 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1601-2) at 1/50,000 dilution and competitor's antibody at 1/10,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Fig3: All lanes: Western blot analysis of HMGB1 with anti-HMGB1 antibody [SA39-03] (ET1601-2) at 1:500 dilution.

Lane 1: Wild-type Raw264.7 whole cell lysate.
Lane 2: HMGB1 knockout Raw264.7 whole cell lysate.

ET1601-2 was shown to specifically react with HMGB1 in wild-type Raw264.7 cells. No band was observed when HMGB1 knockout samples were tested. Wild-type and HMGB1 knockout samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary Anti-HMGB1 antibody (ET1601-2, 1/500) and Anti-HSP90 antibody (ET1605-56, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.

Cell lysate was provided by Ubigene Biosciences (Ubigene Biosciences Co., Ltd., Guangzhou, China).

Fig4: Western blot analysis of HMGB1 on different lysates with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/20,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: HCT 116 cell lysate (20 µg/Lane)
Lane 3: A549 cell lysate (20 µg/Lane)
Lane 4: HepG2 cell lysate (20 µg/Lane)
Lane 5: Jurkat cell lysate (20 µg/Lane)
Lane 6: Mouse thymus tissue lysate (20 µg/Lane)
Lane 7: Rat spleen tissue lysate (30 µg/Lane)

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

Exposure time: 3 minutes 10 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1601-2) at 1/20,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

	Fig5: Immunocytochemistry analysis of MCF-7 cells labeling HMGB1 with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/100 dilution. Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/100 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
	Fig6: Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/20,000 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1601-2) at 1/20,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
	Fig7: Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/20,000 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1601-2) at 1/20,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

	Fig8: Immunohistochemical analysis of paraffin-embedded mouse hippocampus tissue with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/20,000 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1601-2) at 1/20,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
	Fig9: Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/20,000 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1601-2) at 1/20,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
	Fig10: Immunohistochemical analysis of paraffin-embedded rat hippocampus tissue with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/20,000 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1601-2) at 1/20,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Fig11: "Immunocytochemistry analysis of C2C12 cells labeling HMGB1 with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Fig12: Immunocytochemistry analysis of C6 cells labeling HMGB1 with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-HMGB1 antibody (ET1601-2) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Fig13: Flow cytometric analysis of HCT 116 cells labeling HMGB1.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1601-2, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

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