Highly Characterized Antibodies For Use In Several Applications

With all the characterization data published on the Human Protein Atlas (HPA) portal (www.proteinatlas.org), Prestige Antibodies are the most highly characterized antibodies in the industry today. There are over 12,000 Prestige Antibodies available, covering 10,000 human protein targets. This corresponds to approximately 50% of the human proteome. Each year more than 2,500 new Prestige Antibodies are made available to the scientific community. The antibodies are directed against both new and previously unknown proteins as well as established protein families.

Each Prestige Antibody has been analyzed and annotated in a large variety of normal and diseased human tissues and the expression profiles are conveniently searchable online. Immunohistochemical (IHC) staining has been performed in 46 normal human tissues, the 20 most common cancer types, 59 cells and cell lines. In addition, all antibodies have been tested for performance in Immunofluorescence (IF) and Western Blot (WB) applications. In total, more than 700 IHC, as well as ICC, IF and WB images per antibody, are shown online.

The Human Protein Atlas Program is systematically generating antibodies against the human proteome and the resulting Human Protein Atlas provides a useful platform for biomarker discovery efforts.

Malignant Melanoma

Malignant melanoma is a common skin tumor with a rapidly increasing incidence rate. Survival rates are high, but melanomas tend to metastasize relatively early, and for patients with metastatic melanoma prognosis is poor, with a 5-year survival rate of less than 10%. Today, there are no validated biomarkers for use in a clinical setting that are able to give prognostic information and estimate the risk for metastatic disease.

Syntaxin-7

Syntaxin-7 (STX7) was identified on the Human Protein Atlas as a protein showing a selective expression profile in cancer tissue as shown in Figure 1.

Figure 1. Detection of STX7 in HPA screening tissue micro arrays (TMAs). This schematic overview shows the staining results in cancer tissue with the color codes representing different levels of immunoreactivity (red=strong staining, orange=moderate staining, yellow=weak staining, white=negative staining and black=missing or non-representative tissues). Duplicate samples are displayed from each individual.

Syntaxin-7 is a protein that belongs to the SNARE family, proposed to be mediators of all intracellular fusion events. Syntaxin-7 is believed to be a regulator of membrane vesicular trafficking between late endosomes and lysosomes.

The Prestige Antibody HPA001467, directed against Syntaxin-7, was used to evaluate the potential of syntaxin-7 as a marker for malignant melanoma prognosis. The antibody was successfully used in IHC, WB and IF applications. IHC staining was performed on tissue microarrays as well as on two extended melanoma cohorts. As can be seen in Figure 2, there was a clear differentiation in expression in different melanomas. This differential expression could be correlated to tumor aggressiveness. It was shown that high expression of STX7 indicates good prognosis.

Figure 2. Immunohistochemical staining of malignant melanoma using the Anti-STX7 antibody. Strong staining is illustrated in A and negative/weak staining in B.

The expression of syntaxin-7 in two melanoma cell lines was confirmed by Western Blot analysis (Figure 3).

Figure 3. Western Blot analysis of STX7 in the melanoma cell lines SK-MEL-30 (middle lane) and WM-115 (right lane). The left lane shows the molecular weight markers. A strong band within the predicted size range (30 kDa) could be detected in the melanoma cell lines.

Immunofluorescence and confocal microscopy was used to determine the subcellular localization of syntaxin-7 in SK-MEL-30 cells. As shown in Figure 4, vesicles along the plasma membrane with accumulation of vesicles in the juxtanuclear region are stained.

Figure 4. Confocal images of immunofluorescently stained SK-MEL-30 cells. STX7 antibody staining is shown in green, tubulin staining in red and nuclear staining in blue. The microscope settings have been adjusted to visualize the intense staining of vesicles in the outer region of the cytoplasm. Scale bar=10 μm.

Summary

• The Human Protein Atlas was used to identify STX7 as a potential prognostic marker for malignant melanoma.
• It was shown that reduced levels of STX7 are associated with more aggressive tumors.
• The Prestige Antibody HPA001467 has been validated as an excellent tool for biomarker detection.

Use of Prestige Antibodies in Detection Studies

Prestige Antibodies are excellent tools for detection studies. These are highly characterized antibodies evaluated in several applications, such as IHC, IF and WB, and tested on a large variety of normal and diseased human tissues. Standardized universal protocols can be used and all characterization data are conveniently searchable online (www.proteinatlas.org).

Learn more about Prestige Antibodies at sigma.com/prestige.

References

Strömberg S, Agnarsdóttir M, Magnusson K, Rexhepaj E, Bolander A, Lundberg E, Asplund A, Ryan D, Rafferty M, Gallagher WM, Uhlén M, Bergqvist M, Pontén F. (2009) Selective expression of Syntaxin-7 protein in benign melanocytes and malignant melanoma. J Proteome Res. 8(4):1639-46.

Berglund L et al. A gene-centric human protein atlas for expression profiles based on antibodies. Mol Cell Proteomics 2008 (10):2019-27. 9.