Precision extraction of podocin

Basic Introduction to Podocin

Biofilm contains proteins called podocin, is the main bearer of biofilm function. According to the difficulty of protein separation and the location of the distribution in the membrane, podocin can be divided into three categories: external podocin, or peripheral podocin, podocin, or integrated podocin and lipid anchor protein.

Podocin is an important component of the plasma membrane. It plays a very important biological role in accepting the receptor protein of the external signal molecule, the carrier protein and the channel protein carrying the substance transport, the enzyme protein catalyzing various reactions and so on.

Definition of Podocin

Biofilm contains proteins called podocin, is the main bearer of biofilm function.

Podocin is the main manifestation of membrane function. It is estimated that approximately 30% of the proteins encoded by the nuclear genome are podocin. According to the binding of podocin to lipid molecules, they can be divided into integral proteins, peripheral proteins and lipid-anchored proteins.

Classification of Podocin

According to the difficulty of protein separation and the location of the distribution in the membrane, podocin can be divided into two categories: extrinsic protein and the intrinsic podocin or integral protein (intrinsic protein ). The outer Podocin accounts for about 20% to 30% of the podocin, which is distributed on the inner and outer surface of the membrane, mainly on the inner surface. It is a water-soluble protein. It binds to the polar head of membrane molecule through ionic bond and hydrogen bond. , Or through the interaction with the intrinsic protein, indirectly with the membrane binding; intrinsic protein accounts for about 70% to 80% of Podocin, is the amphiphilic media molecules can be embedded in different degrees of lipid bilayer molecules. And some throughout the lipid bilayer, both ends of the membrane exposed to the internal and external surface, this type of Podocin, also known as transpodocin. The inner part of the podocin which is exposed to the membrane contains more polar amino acids, which is hydrophilic and is close to the hydrophilic head of the phospholipid molecule. The Podocin embedded in the lipid bilayer consists of some non-polar amino acids, The hydrophobic tail of the molecule binds to each other and is therefore very tightly bound to the membrane.

Lipid-anchored proteins can be divided into two classes: glycophosphatidylinositol (GPI) -linked proteins, GPI located in the outer lobules of the cell membrane, and phospholipase C (which recognizes inositol-containing Phospholipids) to release the bound protein. Many cell-surface receptors, enzymes, cell adhesion molecules, and PrPC causing scrapie are all such proteins. Another lipid-anchored protein binds to a long hydrocarbon chain inserted into the leaflet of a plasma membrane, such as the [alpha] and [gamma] subunits of the trimeric GTP-binding regulatory protein.

Podocin function

The function of Podocin is multifaceted. Some podocin can act as & quot; carriers & quot; to transport substances into and out of cells. Some Podocin are hormones or other chemical substances specific receptor, such as thyroid cells to accept the pituitary from the thyroid-stimulating hormone receptor. Membrane surface and a variety of enzymes, so that specific chemical reactions can be carried out on the membrane, such as the endoplasmic reticulum membrane can catalyze the synthesis of phospholipids. The cell recognition function is also determined by the protein on the membrane surface. These proteins are often surface antigens. Surface antigen and specific antibodies can be combined, such as human cell surface has a protein antigen HLA, is a highly variable dimer. Different people have different HLA molecules, organ transplantation, the implanted organs are often excluded, it is because the implantation of HLA molecules are not accepted by the recipient of the reason.

To summarize, Podocin function as 1) transporters; 2) enzymes; 3) receive recognition signals; 4) link structures.

Mobility of Podocin

Many Podocin in the lipid bilayer can move freely, which can be through the human and mouse fusion of two cells, and then one of the antibodies to cells, such as mouse antibody to fluorescent dye staining, and then this Antibodies with fluorescence are placed in the outer media of the fused cells and the antibody binds to the surface antigens of the mouse cells. Results The cell surface of the mice also had fluorescence, while the human cells did not fluoresce due to the mismatch of mouse antibody binding. So that you can distinguish the two kinds of cell surface antigen under fluorescent microscope. The results showed that the surface antigens of human and murine cells were distinctly different from each other when the cells began to fuse. But after fusion, the two antigens gradually distributed evenly on the surface of the fusion cells. This experiment demonstrates that the Podocin is mobile and also demonstrates membrane fluidity.

But not all podocin can move in the membrane, some podocin can not be moved or can not move freely.

Podocin extraction

Extraction of podocin from fresh samples (simplified method)

(PH 8.5-9.0): 50mM Tris-HCl, 2mM EDTA, 100mM NaCl, 0.5% Triton X-100, pH adjusted to 8.5-9.0. Use 100μg/ml prior to lysis Lysozyme, 1 [mu] l /ml protease inhibitor PMSF. The amount of the lysate was 10-50 ml of lysis buffer/1 g of wet cells.

2, 40ml of bacteria in 12000g, 4°C centrifuge 15 minutes to collect the bacteria, the precipitate suspended in PBS washed 2 times, 1ml lysis suspension by adding cell suspension.

3, ultrasonic crushing, using 300w, 10s ultrasound/10s interval, ultrasound 20min, repeated freeze-thaw ultrasound 3 times to clear or discoloration of bacteria.

4,1000g centrifuged to remove large debris, supernatant can be directly denatured PAGE electrophoresis, or 1% SDS solution after dialysis frozen.