CH 3 Flashcards
MSG in foods
Commonly associated with:
• Chinese food, Soy sauce, Yeast extract, GRAS (FDA)
• Grandfathered in before 1958
• Sensitive people (within mins):
- Headache, sweating, rapid breathing, tightness in
chest
• With IBS: headache, diarrhea, GI pain
and bloating, fatigue, muscle pain,
and cognitive dysfunctionUmami
The “savory” flavor
• Walnuts, fish broth
• High levels of glutamate are found in foods like soy sauce and parmesan cheese
• Excitatory neurons in the tongue are stimulated
- T1R1/T1R3 Receptor in tongue
• Too much can lead to neurotoxicity – overexcited to
death
Purification
• In order to study proteins in vitro, they must be isolated from other components. In essence, this is hard because of how many proteins exist within a give biological sample
- Separation can also result in inactivation of the protein - this would be a problem because the protein can lose its function
What intrinsic characteristics can be used to tease out specific proteins?
Size, charge, solubility, binding specificities
Ammonium Sulfate
- separation by solubility
- crude operation is often the first step
- salting out takes advantage of the solubility of proteins in salt solutions
- you can precipitate out impurities first providing a purer sample in the supernatant
- eventually you gather your precipitate of interest and re-suspend it in small amounts of solvent
Salting out by ammonium sulfate
1) in a test tube, pour starting volume of sample plus pH-adjusting buffer. pour in saturated or subsaturated ammonium sulfate
2) mix together
3) ammonium sulfate concentration is increased ([salt] increased = water molecules attracted to the salt, decreasing the number of water molecules available to interact with charged part of protein)
4) visible protein precipitates (protein molecules coagulate by forming hydrophobic interactions with each other)
5) salting out process is completed
Column Chromatography
- used to purify compounds depending on their polarity or hydrophobicity/ solubility
- often used to separate a mixture of proteins through an insoluble matrix
- proteins are separated as they interact and run through the matrix when solvent is added and thus divided into fractions
- we measure absorbance at 280 nm because of amino acids with aromatic rings. At this wavelength, the absorption of proteins is mainly due to the amino acids tryptophan, tyrosine and cysteine with their molar absorption coefficients decreasing in that order.
High-Performance Liquid Chromatography
- column chromatography performed under high pressure using small, tightly packed columns with solvent flow controlled by computer
Ion Exchange Chromatography
- separates ions and polar molecules based on their affinity to the ion exchanger
- the column contains positive charges (anion-exchange) or negative charges (cation-exchange) resins on the surface
- What will that do to proteins being passed through the column? It will trap oppositely charged proteins that can be salted out and isolated
- What would you need to know about your protein of interest here? Binding specificity
Ion exchange chromatography Principle
- positively charged proteins bind to negatively charged beads (negative proteins flow through/ filter out)
- negatively charged proteins bind to positively charged beads (positive proteins flow through/ filter out)
Gel filtration chromatography
- separates proteins on the basis of size
- gel is made out of porous beads that can trap smaller proteins
- larger molecules travel faster and can be eluted faster
1) very small molecules enter many pores in the gel, equilibrating between the gel and the moving buffer, and so travel slowly and are diluted later
2) medium sized molecules enter some pores in the gel, equilibrating between the gel and the moving buffer
3) large molecules enter few pores in the gel and so travel rapidly and are eluted sooner
Affinity chromatography
- one of the newer techniques
- the most selective
- uses very specific binding of target protein and other molecule bound to the column (e.g. and antibody)
- target protein can be eluted out
1) the proteins specifically bind to the column
2) the unbound proteins are washed away
3) the specifically bound proteins are eluded
SDS-PAGE (Western Blot Apparatus)
This device operates on the principal that denatured protein samples can be run through a polyacrylamide matrix and can be separated by size and charge.
- The buffer is alkaline to promote anionic charge and thus being drawn down the column towards the positive charge anode ( - to +)
• Sodium dodecyl sulfate
polyacrylamide gel electrophoresis
Polyacrylamide Gel
Once run completely
through the gel, the dye-edge is typically allowed to run-off and the gel can be used for
separation of protein or
for analysis
• Antibodies are used to detect specific proteins on the gel, differentiating them from the others
Horse Radish Peroxidase Reaction
- HRP is often conjugated to an antibody to detect a target molecule: the antibody specifically locates the target, and HRP, in company with a substrate, produces a detectable signal.
1) blocking reagent blocks unoccupied sites on the membrane
2) primary antibody to a specific antigen is incubated with the membrane
3) a blotting grade antibody-enzyme conjugate is added to bind to the primary antibody
4) substrate reagent is then added to the blotting-grade antibody enzyme
5) the enzyme catalyzes the substrate (S) to form a detectable product (P) at the site of the antigen-antibody complex
