Detection of Cathepsins in Cytoplasm

Background of Cathepsins Detection

Lysosomes contain more than 50 different acid hydrolases that exhibit optimal activity at relatively low pH values (4-5). The lysosomal membrane is an important barrier for lysosomes and the most abundant proteins are highly glycosylated transmembrane proteins that protect the membrane from degradation of lysosomal enzymes. Involved in lysosomal membrane permeability (LMP) is membrane damage induced by a large number of cytotoxic stimuli under a variety of cellular conditions culminating in cell death.The distinguishing feature of LMP is the translocation of soluble hydrolases in the lumen to cytoplasmic lysates. The partial and selective release of these histones (such as histones B and D) triggers a series of cellular signaling events that lead to cell death. In many cases, cell death induced by LMP can be partially inhibited by histone inhibitors.

Fig. 1. Secretagogue induced AP results in release of Cathepsin-B and other lysosomal-enzymes into the cytosol. (Talukdar R, et al., 2016)

Cathepsins in Cytoplasm Detection Services

Traditionally lysosomal membrane permeability has been assessed using a β-glycerophosphate substrate, but this substrate readily penetrates the lysosomal membrane only under conditions of altered permeability. CD BioSciences offers multiple methods to quantify and visualize lysosomal permeability during cell death. Here, we performed a visual analysis of lysosomal proteases released from cytoplasmic lysates to assess LMP.

We developed a detailed protocol for immunocytochemical staining based on the lysosomal protease cathepsin L, which can estimate the extent of LMP by simultaneously measuring changes in several different sizes of cathepsin over time.

1. Cells are cultured and lysosomal membrane damage is caused by cytotoxic agents (compounds or siRNA).
2. Immunocytochemical immunostaining of cells.
3. Observe cells by fluorescence or confocal microscopy.
4. Cells were imaged to analyze vesicular and diffuse staining of lysosomal cathepsin L and the absence and presence of active Bax.

Characteristics of This Approach

• Histone proteases released during LMP produce diffuse fluorescence patterns throughout the cell.
• The level of histone activity can be normalized to the protein level by using a protein quantification kit.
• Histases can be detected in cytosolic extracts using histidine-specific substrates and a fluorescent enzyme marker.
• Cytosolic release of histone enzymes can be detected using protein blotting.
• Histoplasma-positive cells in tissue sections can be counted using fluorescence microscopy.
• The most commonly used method is to detect the expression of histones B and D.
• Immunocytochemical immunostaining procedures can be performed within 4-5 hours.

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Reference

1. Talukdar R, Sareen A, et al. (2016) Release of Cathepsin B in Cytosol Causes Cell Death in Acute Pancreatitis. Gastroenterology. 151(4):747-758.e5.