53BP1 (HMV324)

p53-binding protein 1 (53BP1) is a scaffold protein without enzymatic activity which is encoded by the TP53BP1 gene at chromosome 15q15.3. 53BP1 participates in DNA damage checkpoint control and DNA repair, and acts as a key determinant of DNA double-strand break (DSB) repair pathway choice. The main mechanisms for DSB repair include the homologous recombinational repair (HR) pathway (requiring the sister chromatid as a template) and the non-homologous end-joining (NHEJ) pathway which rejoins the broken ends without the use of extensive homology. NHEJ, although faster than HR, is less accurate. The early divergent step between these two pathways is end resection which is regulated by numerous factors including BRCA1 and 53BP1. In response to DSBs, 53BP1 rapidly accumulates on the chromatin surrounding the DNA damage site  and recruits the DNA break-responsive effectors that promote NHEJ-mediated DSB repair while inhibiting homologous recombination (HR) signaling. Reduced function of 53BP1 may promote the even more error-prone microhomology-mediated end joining (MMEJ), also known as alternative nonhomologous end-joining (Alt-NHEJ) which normally is responsible for less than 10% of DSB repair and plays a major role in aging, genomic instability and cancer. Loss of p53BP1 results in resistance to PARP inhibition treatment in Brca1-deficient tumors.

A nuclear 53BP1 staining occurs in virtually all tissues and cell types. The level of 53BP1 expression varies between tissues/cell types to some extent. It is particularly low or absent in hepatocytes. In the testis, the 53BP1 staining is strong in spermatogonia but staining intensity gradually decreases with maturation of germ cells. Mature spermatocytes and spermatids are 53BP1 negative.  Images describing the 53BP1 staining pattern in normal tissues obtained by the antibody HMV324 are shown in our “Normal Tissue Gallery”.

These findings are largely comparable to the RNA and protein data described in the Human Protein Atlas (Tissue expression 53BP1).

Positive control = Testis: 53BP1 staining should be strong in spermatogonia but decrease with maturation of germ cells.

Negative control = Testis: 53BP1 staining should be absent in mature spermatocytes and spermatids.

Normal tissue gallery

Cancer tissue gallery

No data available at the moment

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 HMV324 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.

• The role of 53BP1 in DNA repair needs to be further investigated.
• The prognostic and predictive role of 53BP1 expression levels in cancer is unclear.

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 HMV324 specificity is in line 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 53BP1). In agreement with HMV324 immunostaining data, 53BP1 RNA expression predominated in the  brain and in lymphoid tissues and it was comparably low in the liver. However, orthogonal validation is not optimal for assessing ubiquitously expressed protein.

Comparison of antibodies: True expression of 53BP1 in all cell types with 53BP1 positivity seen by HMV324 is corroborated by an identical staining obtained by a commercially available independent second antibody (termed “validation antibody”). Most of all, both antibodies showed a complete lack of nuclear staining only in mature spermatocytes and spermatids of the testis, and in hepatocytes in the liver. Moreover, both antibodies showed a particularly low staining intensity of epithelial cells of villi of the small intestine, and a particularly strong staining of islet cells of the pancreas.

Normal tissue gallery