what is Electrophoresis
Electrophoresis is a method commonly used to separate charged molecules such as proteins and nucleic acids. The basic principle is that charged molecules will migrate through a liquid or semisolid medium that is subjected to an electric field.
What is SDS-PAGE
SDS-PAGE = Sodium dodecyl sulfate polyarylamide gel
How do charged molecules move in electrophoresis
Negatively charged molecules will migrate towards the positive pole (anode) and conversely, positively charged molecules will migrate towards the negative pole (cathode). Molecules that differ in charge and size will migrate to form distinct bands in a gel matrix. These bands can be compared to a standard of proteins of known molecular weight.
What is the electrophoresis chamber
The electrophoresis chamber is such that it holds the cast gel in a buffered solution with power electrodes present in the buffered solutions on either end of the gel. The power supply unit converts AC of the line source to DC which is required for electrophoresis. A specific voltage can be selected, depending on the type of gel. Since the only electrical connection between the electrodes is the buffer in the gel, the charged molecules will migrate through the gel.
what types of gels are used in electrophoresis
Many types of gels can be used for electrophoresis such as agarose, acrylamide, cellulose acetate or starch. Each their advantages.
what is the most commonly used method to separate proteins
The most commonly used method to separate proteins is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In this gel matrix, acrylamide is cross-linked with N,N’ methylene-bis-acrylamide to form a gel matrix.
what is sodium dodecyl sulfate
Sodium dodecyl sulfate is a detergent that denatures and binds to proteins leading to an overall negative charge. It is known that 1.4g of SDS will bind 1g of protein, thus SDS-protein complexes will migrate as though they have the same charge to mass ratio.
what determines the mobility of the proteins
When the charge-to-mass ratio is the same, the mobility of the proteins is related to the molecular weight because of the molecular sieving properties of the gel.
why do we need a tracking dye
Proteins run in gels are invisible and can only be tracked in the gel with the use of tracking dye, often bromophenol blue, which is added to the sample buffer.
How does the dye moves in electrophoresis
During electrophoresis the tracking dye moves just ahead of the fastest running proteins.
What does the sample buffer do
The sample buffer, which is mixed with the protein samples, contains SDS, tracking dye, Beta-mercaptoethanol and sucrose or glycerol, whose density prevents the sample from mixing with the running buffer in the Tetra cell unit while the gel is being loaded.
What does Beta-mercaptoethanol do
Beta-mercaptoethanol is mixed with the protein samples to dissociate multichain proteins by reducing disulfide linkages. This allows the proteins to bind to SDS and assume a random coil configuration. Proteins bound to SDS will separate based on molecular weight.
What is the protein ladder
A set of protein standards, or proteins of known molecular weights, are run alongside the samples to determine the molecular weights of the protein samples. These protein standards, also known as a protein ladder, contain purified proteins of known molecular weights.
how do we determine the molecular weights of the proteins of interest
A standard curve of the log of the molecular weights of the protein standard can be plotted against the distance traveled in the gel in order to precisely determine the molecular weights of the proteins of interest.
What does staining do
Proteins are not visible in a gel without staining them with a dye. for direct visualization of proteins in the SDS-PAGE gel, several dyes are known to irreversibly bind to amino acids of proteins, but not to the gel matrix. Coomassie brilliant blue is a common choice for protein staining, but silver staining is an alternative.
what does Coomassie brilliant blue dye do
Coomassie brilliant blue dye reacts and binds to arginine and to aromatic amino acids, such as tyrosine, phenylalanine, and tryptophan residues revealing the location of the protein bands in the gel.
why do we need to de-stain the gel
The dye will initially stain the entire gel, so in order to resolve the distinct protein bands, the gel must be de-stained. The dye will remain bound to the proteins but will be washed away from the gel.
How can we detect a specific protein
In order to detect specific proteins a technique called Western blotting can utilize antibodies to detect specific proteins. Proteins are transferred to a nitrocellulose membrane after gel electrophoresis is complete. A specific protein can be detected with a primary antibody to the protein. A secondary antibody conjugate to an enzyme (C-reactive protein or horse radish peroxidase) will recognize the primary antibody and in the presence of the enzymes’ substrate will give off light detectable on X-ray film. Proteins can also be radioactively labeled which allows for direct detection on X-ray film.
what are the two layers to SDS-Page
There are two layers to SDS-PAGE gels:
- Stacking layer
- Resolving layer
what is the stacking layer made from
The stacking layer is made using a Tris buffer pH 6.8 and with a low percentage of acrylamide (~4%). This allows the samples to condense into a layer before being separated in the resolving layer.
what is resolving layer made of
The resolving layer consists of a Tris buffer pH 8.8 and a higher concentration of acrylamide (8-15%). The higher the concentration of acrylamide the higher the resolution is. This is ideal for proteins of similar or small molecular weights. For lab 7 we use a 12% acrylamide resolving gel which is suitable for 14.4-97.4kDa
what is 2X sample buffer
2X sample buffer is made from 62.5mM Tris-HCl, pH 6.8, 25% Glycerol, 2% SDS, 0.01% Bromophenol blue, 0.5% Beta-mercapotoethanl
what does each part of 2X sample buffer do
The SDS denatures the protein and adds a negative charge, the Beta-mercaptoethanol breaks disulfife bonds, the glycerol increases density to ensure sinking in the well, bromophenol blue acts as a tracking dye, Tris maintains a sable pH for the SDS-PAGE system.
what is 1X running buffer
1X running buffer is made from 24.7 mM Tris base, 19 mM glycine, 0.01% SDS
