Product details
Synonyms = xxx
Antibody type = Recombinant Rabbit monoclonal / IgG
Clone = HMV317
Positive control = Colon: A strong HMGB1 positivity should be seen in all cell types.
Negative control =Testis: Spermatids and more mature spermatocytes should be HMGB1 negative.
Cellular localization =Intracellular
Reactivity = Human
Application = Immunohistochemistry
Dilution = 1:100 – 1:200
Intended Use = Research Use Only
Relevance of Antibody
HMGB1 is a multifunctional nuclear protein also acts as a damage-associated molecular pattern molecule (DAMP) in the extracellular space.
Biology Behind
High-mobility group protein B1 (HMGB1) is a protein consisting of 215 amino acid residues which is coded by the HMGB1 gene on chromosome 13q12.3. High-Mobility Group (HMG) chromosomal proteins are the most significant nuclear non-histone proteins in humans. As all other members of the non-histone chromosomal high-mobility group protein family, HMGB1 is a chromatin-associated protein which is ubiquitously distributed in human cells. HMGB1 is the second most abundant protein (after histone) inside the nucleus. It exerts multiple functions in the nucleus, the cytoplasm, and in the extracellular space. In the nucleus, HMGB1 plays a role in the maintenance of nucleosome structure, as well as the regulation of DNA replication, transcription, and repair. HMGB1 increases the binding affinity of many transcription factors (for example: p53, Rb, NF-κB, estrogen receptor) to their target DNA sequences. Probably due to its ability to specifically bind to damaged DNA sequences, HMGB1 contributes to the early stage of DNA repair. In the extracellular space, HMGB1 acts as a damage-associated molecular pattern molecule (DAMP) that mediates inflammation and immune responses. In case of tissue damage, HMGB1 is released through active secretion of HMGB1 from monocytes, natural killer cells, dendritic cells (DC), platelets, and endothelial cells or by passive release from the nuclei of necrotic cells. Extracellular HMGB1 is thought to contribute to various conditions, including sepsis, atherosclerosis, arthritis, neurodegeneration, meningitis, and cancer. Therapeutic options to regulate HMGB1 in preclinical models are being evaluated.
Suggested reading: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104204/
Staining Pattern in Normal Tissues
Images describing the Caspase-3 staining pattern in normal tissues obtained by the antibody HMV317 are shown in our “Normal Tissue Gallery”.
| Brain | Cerebrum | Nuclear HMGB1 staining occurs primarily in glia cells and in vessels. Neurons are largely HMGB1 negative. |
| Cerebellum | Nuclear HMGB1 staining occurs in glia cells and it is strongest in granule cells. Purkinje cells are largely negative. | |
| Endocrine Tissues | Thyroid | Significant nuclear HMGB1 staining of all cells. |
| Parathyroid | Strong nuclear HMGB1 staining of all cells. | |
| Adrenal gland | Significant nuclear HMGB1 staining of all cells. | |
| Pituitary gland | Epithelial cells of the adenohypophysis are largely negative. In the neurohypophysis, pituicytes only show a faint HMGB1 positivity. | |
| Respiratory system | Respiratory epithelium | Significant nuclear HMGB1 staining of all cells. Among epithelia, HMGB1 staining is most intense in basal cells. |
| Lung | Strong nuclear HMGB1 staining of all cells. | |
| Gastrointestinal Tract | Salivary glands | Significant nuclear HMGB1 staining of all cells. |
| Esophagus | HMGB1 staining intensity is strongest in the basal and suprabasal cell layers and continuously decreases towards the superficial cell layers. | |
| Stomach | Significant nuclear HMGB1 staining of all cells. | |
| Duodenum | Significant nuclear HMGB1 staining of all epithelial and inflammatory cells. HMGB1 staining is somewhat weaker in Brunner glands. | |
| Small intestine | Significant nuclear HMGB1 staining of all cells. | |
| Appendix | Significant nuclear HMGB1 staining of all cells. | |
| Colon | Significant nuclear HMGB1 staining of all cells. Staining intensity is higher in the crypt base than in the surface epithelium. | |
| Rectum | Significant nuclear HMGB1 staining of all cells. Staining intensity is higher in the crypt base than in the surface epithelium. | |
| Liver | Significant nuclear HMGB1 staining of all cells. Staining is particularly high in bile ducts and rather low in hepatocytes. | |
| Gallbladder | Significant nuclear HMGB1 staining of all cells. | |
| Pancreas | Significant nuclear HMGB1 staining of all cells. | |
| Genitourinary | Kidney | Significant nuclear HMGB1 staining of all cells. Staining is particularly low in tubuli (especially proximal) and highest in collecting ducts and glomeruli. |
| Urothelium | Strong nuclear HMGB1 staining of all urothelial cell layers. | |
| Male genital | Prostate | Significant nuclear HMGB1 staining of all cells. Staining is particularly low in acinar cells and highest in basal cells. |
| Seminal vesicles | Significant nuclear HMGB1 staining of all cells. Among epithelial cells, HMGB1 staining is more intense in basal than in luminal cells. | |
| Testis | Significant nuclear HMGB1 staining of Sertoli and Leydig cells. HMGB1 staining is weaker in the germ cells where the staining level gradually decreases with maturation from spermatogonia to spermatocytes and spermatids (which are negative). | |
| Epididymis | Significant nuclear HMGB1 staining of all cells. In the corpus, HMGB1 staining is more intense in basal than in chief cells. | |
| Female genital | Breast | Significant nuclear HMGB1 staining of all cells. HMGB1 positivity is strongest in basal and luminal epithelial cells. |
| Uterus, myometrium | Significant nuclear HMGB1 staining of all cells. | |
| Uterus, ectocervix | HMGB1 staining intensity is strongest in the basal and suprabasal cell layers and continuously decreases towards the superficial cell layers. | |
| Uterus endocervix | Significant nuclear HMGB1 staining of all cells. | |
| Uterus, endometrium | Strong nuclear HMGB1 staining of epithelial and stromal cells. Staining of epithelial cells may be markedly less intense during the secretion phase. | |
| Fallopian Tube | Strong nuclear HMGB1 staining of all cells. | |
| Ovary | Strong nuclear HMGB1 staining of stromal cells but HMGB1 staining is only faint in the corpus luteum. | |
| Placenta early | Significant nuclear HMGB1 staining of all cells. | |
| Placenta mature | Significant nuclear HMGB1 staining of all cells. | |
| Amnion | Moderate to strong nuclear HMGB1 staining of amnion cells. | |
| Chorion | Strong nuclear HMGB1 staining of chorion cells. | |
| Skin | Epidermis | HMGB1 staining intensity is strongest in the basal and suprabasal cell layers. It continuously decreases towards the superficial cell layers. |
| Sebaceous glands | Significant nuclear HMGB1 staining of all cells. | |
| Muscle/connective tissue | Heart muscle | Nuclear HMGB1 staining is only weak in heart muscle cells. |
| Skeletal muscle | Strong nuclear HMGB1 staining. | |
| Smooth muscle | Significant nuclear HMGB1 staining. | |
| Vessel walls | Significant nuclear HMGB1 staining. | |
| Fat | Strong nuclear HMGB1 staining. | |
| Stroma | Significant nuclear HMGB1 staining. | |
| Endothelium | Strong nuclear HMGB1 staining. | |
| Bone marrow/ lymphoid tissue | Bone marrow | Intense nuclear HMGB1 staining of virtually all cells of the hematopesis. |
| Lymph node | Strong nuclear HMGB1 staining of virtually all cells of the immune system. | |
| Spleen | Strong nuclear HMGB1 staining of virtually all cells of the immune system. | |
| Thymus | Strong nuclear HMGB1 staining of virtually all cells of the immune system. HMBG1 labeling was weaker in corpuscles of Hassall’s, especially in their central areas. | |
| Tonsil | Strong nuclear HMGB1 staining of virtually all cells of the immune system. A significant HMGB1 staining also occurs in the squamous epithelium where the staining intensity continuously decreases towards the top layers. | |
| Remarks |
A nuclear HMGB1 expression occurs in virtually all tissues and cell types. The level of HMGB1 expression varies between tissues/cell types to some extent. A particularly low level of HMGB1 expression occurs in maturing spermatocytes and spermatids of the normal testis and in epithelial cells of the adenohypophysis. RNA and protein expression data of HMGB1 findings are also described in the Human Protein Atlas (Tissue expression HMGB1).
Positive control = Colon: A strong HMGB1 positivity should be seen in all cell types.
Negative control =Testis: Spermatids and more mature spermatocytes should be HMGB1 negative.
Staining Pattern in Relevant Tumor Types
A positive HMGB1 immunostaining (at variable levels of intensity) can be seen in tumor cells of virtually all cancer types as well as in tumor associated stromal and inflammatory cells.
The TCGA findings on HMGB1 RNA expression in different tumor categories have been summarized in the Human Protein Atlas.
Compatibility of Antibodies
No data available at the moment
Protocol Recommendations
IHC users have different preferences on how the stains should look like. Some prefer high staining intensity of the target stain and even accept some background. Others favor absolute specificity and lighter target stains. Factors that invariably lead to more intense staining include higher concentration of the antibody and visualization tools, longer incubation time, higher temperature during incubation, higher temperature and longer duration of the heat induced epitope retrieval (slide pretreatment). The impact of the pH during slide pretreatment has variable effects and depends on the antibody and the target protein.
All images and data shown here and in our image galleries are obtained by the manual protocol described below. Other protocols resulting in equivalent staining are described as well.
Manual protocol
Freshly cut sections should be used (less than 10 days between cutting and staining). Heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7,8 Target Retrieval Solution buffer. Apply HMV317 at a dilution of 1:200 at 37°C for 60 minutes. Visualization of bound antibody by the EnVision Kit (Dako, Agilent) according to the manufacturer’s directions.
Potential Research Applications
- The clinical significance (prognostic/predictive) of HMGB1 expression levels in cancer is unknown.
- The exact mechanism of HMGB1’s translocation from the nucleus to cytoplasm then out into the extracellular matrix is not fully understood.
Evidence for Antibody Specificity in IHC
There are two ways how the specificity of antibodies can be documented for immunohistochemistry on formalin fixed tissues. These are: 1. Comparison with a second independent method for target expression measurement across a large number of different tissue types (orthogonal strategy), and 2. Comparison with one or several independent antibodies for the same target and showing that all positive staining results are also seen with other antibodies for the same target (independent antibody strategy).
Orthogonal validation: For the antibody HMV317 specificity is suggested by the good concordance of the immunostaining data with data from three independent RNA screening studies, including the Human Protein Atlas (HPA) RNA-seq tissue dataset, the FANTOM5 project, and the Genotype-Tissue Expression (GTEx) project, which are all summarized in the Human Protein Atlas (Tissue expression HMGB1). In agreement with HMV317 immunostaining data, RNA expression predominated in the bone marrow and lymphoid tissues and it was comparably low in the testis. However, it must be understood that orthogonal validation is not optimal for assessing ubiquitously expressed proteins.
Comparison of antibodies: True expression of HMGB1in all cell types with HMGB1 positivity by HMV317 is corroborated by an identical staining obtained by a commercially available independent second antibody (termed “validation antibody”). Most of all, both antibodies showed a decrease of the staining intensity from basal to superficial cell layers in squamous epithelium and complete absence of HMGB1 in only one cell type – maturing spermatids and spermatocytes.
