KAP1 / TRIM28 (HMV335)

Tripartite motif-containing 28 (TRIM28), also termed KAP1 (KRAB-associated protein-1), or transcriptional mediator 1b (TIF1b) is a regulatory protein coded by the TRIM28 gene at chromosome 19q13.43. The protein plays a role in transcription regulation by interaction with the Krüppel-associated box repression domain found in many transcription factors and binding to specific chromatin regions. In addition to regulating gene transcription, KAP1 has a variety of regulatory intracellular functions, such as response to DNA damage, maintaining stem cell pluripotency, cellular differentiation and proliferation, viral suppression, and apoptosis. KAP1 is ubiquitously expressed, and its function depends on posttranscriptional modification.

In normal tissues, TRIM28 is ubiquitously seen in nuclei of all tissues.

Images describing the KAP1 / TRIM28 staining pattern in normal tissues obtained by the antibody HMV335 are shown in our “Normal Tissue Gallery”.

These findings are largely consistent with the RNA data described in the Human Protein Atlas (Tissue expression KAP1 / TRIM28). 

Positive control = Colon: A strong nuclear TRIM28are staining should be seen in all cells.

Negative control = Testis: Spermatids are TRIM28 negative while all other cells show a strong nuclear TRIM28 staining of all cells.

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 HMV335 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 TRIM28 in cancer is not sufficiently understood.
• The role of TRIM28 in viral diseases needs to be further investigated.
• The role of TRIM28 in transcription regulation awaits further clarification.

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 HMV335, specificity of its nuclear staining is consistent 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 KAP1 / TRIM28). In agreement with HMV335 immunostaining data, TRIM28 expression is ubiquitous and somehow predominated in the bone marrow and lymphoid tissues. However, orthogonal validation is not optimal for assessing ubiquitously expressed proteins.

Comparison of antibodies: True expression of TRIM28 in all cell types with nuclear TRIM28 positivity by HMV335 is corroborated by an identical nuclear staining obtained by a commercially available independent second antibody (termed “validation antibody”). Most of all, both antibodies showed absent or reduced saining in hepatocytes of some liver samples.

Normal tissue gallery