Our techniques and expertise

Biochemistry:

• Isolation and purification of cellular compartments and organelles for proteomic and functional studies (e.g. plasma membrane, PAM fraction, mitochondria, MAM fraction, microsomes, Golgi, Lysosomes, nuclei, cytosolic fraction )

Metabolism

• metabolic profiling

Mitochondria

• Isolation of functional mitochondria from different cell lines and tissues

• Subfractionation of mitochondria -  the outer and inner mitochondrial membranes, matrix and mitochondrial intramembrane space

• Evaluation of the integrity and activity of individual respiratory chain complexes

• Evaluation of the mitochondrial bioenergetic parameters 

  • oxygen consumption (Clark electrode)

  • mitochondrial membrane potential (spectrophotometric and spectrofluorimetric)

  • ATP/ADP level, NADH/NAD redox state

  •  intramitochondrial changes of pH

  • mitochondrial swelling

  • mitochondrial ROS production (not only mitochondrial ROS !)

  • activity of mitochondrial transporters (transport of radioactive substrates) 

  • Purification of mitochondrial transporters like  ANT or VDAC on celite and hydroxyapatite column – and reconstitution in to the artificial phospholipids vesicles – measurement of the transport activity

Oxidative stress

•  Manifestation of the oxidative stress in cells and in tissues

  • evaluation of oxidative damage to proteins, lipids and DNA

  • evaluation of the antioxidant status (level of different antioxidant enzymes; activity of CAT, SOD; GSH/GSSG)

Mitochondrial Permeability Transition Pore

• Purification and reconstitution of the mitochondrial permeability transition pore (mPTP) in to the artificial vesicles and measurement its opening

• Evaluation of the mPTP opening in intact cells with the use of radioactive deoxyglucose 

• Isolation of mitochondrial contact sites (DEAE- cellulose column) – complex between ANT, VDAC, HK, CK, CypD, cyt. c 

And many others techniques like:

• Measurement of calcium concentration (level/kinetic of changes) in mitochondria and intact cells using fluorescent dyes

• Measurement of SERCA activity – uptake of calcium in to the purified ER vesicles – using spectrofluorimeter

• In Gel Activity Assay (after Blue Native electrophoresis) – measurement of the activity of the individual mitochondrial respiratory chain complexes and ATP-ase activity of the ATP synthase

• Measurement of the activity of many cellular enzymes (spectrophotometer and fluorimeter)

• Western blotting 

• Acrylamide Blue Native and Clear Native electrophoresis (100kDa – 2 MDa)

• Agarose Blu Native Electrophoresis (300kDa – 10/15 MDa) 

• Co-immunoprecipitation and pull-down experiments

Animal models and cells:

Induction of NAFLD with the use of high fat and high sugar diet in mice

Induction of oxidative stress and hyperthyroid/hypothyroid state in animal models

Induction of diabetes in animal models

Animal surgery

Dissection of organs and tissues

Culturing of various cell lines

Experience in preparation of primary fibroblast cultures from human skin biopsy or animal material