Protein Analysis

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis is a technique that is used to separate proteins in a mixture for the purpose of identifying them individually. Proteins coated in SDS (which gives them a negative charge), are pulled through a thin gel by an electrical current. They separate on the gel based on size--the larger the protein, the slower it moves. Proteins can be stained with Coomassie Blue Dye for visualization or they can be detected by the Western Blotting technique.

Western Immunoblot and ELISA

Western Immunoblot analysis allows researchers to determine the molecular weight of a protein and measure relative amounts of a protein present in different samples. The process consists of the following steps:

1. The proteins are separated via SDS-PAGE.
2. The proteins are transferred, or blotted, onto nitrocellulose paper or a membrane, retaining the same placement as on the gel.
3. The blot is incubated with a generic protein to bind the remainder of the paper. Then, an antibody that specifically binds to a particular protein is added to the solution. This antibody will have an enzyme attached to it that can not be detected at this time.
4. A colorless substrate will then be added that the enzyme attached to the antibody can convert to a colored product, thus revealing the location of the antibody-conjugated protein of interest.

Western Immunoblot Overview

ELISA, Enzyme-Linked Immunosorbent Assay, is very similar to the Western Immunoblot analysis, but allows for more sensitive and effortless quantification of the amount of protein in solution. One type of ELISA is the “sandwich ELISA” where the antigen (or protein to be detected) is sandwiched between two antibodies that recognize different epitopes of the antigen.

1. Antibodies against protein “X” are bound to the bottom of a well
2. A cell lysate is added to the well which may contain protein “X”
3. An enzyme-linked secondary antibody is added
4. A colorless substrate is added which is metabolized by the enzyme
5. There is a color change

The amount of color is quantified by a microplate reader. The greater the color change, the more the protein.

Zymography Zymography is a technique that analyzes enzymatic activity of matrix metalloproteinases (MMPs). Various cell surface-associated proteases are electrophoretically separated under denaturing conditions on a combined polyacrylamide-gelatin gel. After renaturation, the MMPs are ezymatically active and able to digest the gelatin, leaving a clear zone in the stained gel. We are using a modified fluorescent microplate assay similar to Zymography to characterize MMP activity in vocal fold tissue.

Microscopy

To characterize the subcellular localization and relative abundance of proteins involved in laryngeal function, we are using a combination of immunochemical staining and fluorescent fusion protein techniques. Using standard immunofluorescent confocal microscopy, we are able to detect and label proteins in situ (in the cell). In this way, we are able to obtain a better picture of how these proteins localize and interact in their native environment. We are using this approach to study protein behavior in cells before and after vibration. To stain these cells, however, they are usually fixed to a cover slide. This kills the cells in the process.

Indirect confocal fluorescent immunocytochemical staining of human neural stem cells (hNPCs) . NuMA also shows a double focus pattern in mitotic hNPCs. A: Aggregate of hNPCs grown in GM stained with anti- -tubulin (red) and anti-NuMA (green). Note the mitotic cell in the center of the neurosphere showing double foci of NuMA at the spindle poles of the mitotic cell (metaphase chromosomes exclude stain and appear black). B: Adherent hNPCs stained as in (A) showing similar localization of NuMA and -tubulin, C, D: Adherent hNPCs stained with anti-NuMA (red) and anti-nestin (green). Scale bars = 10 m.

To view the protein in live cells, we are using an exciting technique which fuses a fluorescent protein to the end of a protein of interest. Hence, the protein can be tracked in a live cell. Using bacterial cloning and mammalian transfection, a gene encoding a given protein “X” is fused to a gene encoding a fluorescent protein such as green fluorescent protein (GFP), yellow fluorescent protein (YFP) or cyan fluorescent protein (CFP). The GFP gene is found naturally in the Pacific jellyfish, Aequoria Victoria. The fused genes are introduced into laryngeal cells and then expressed. Using fluorescent video-microscopy, we are able to excite the fluorescent protein, and since it is not naturally found in mammalian cells, wherever we see the fluorescent protein we know protein “X” is there also—remember they are fused. Thus, we can observe the subcellular localization of protein “X” in live cells.

Click here to view a timelapse of live cells
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