lab 2 Flashcards by layla gobeil

what is amino acid chromatography?

separation of amino acids with chromatography paper

How well did you know this?

Not at all

Perfectly

by what property does paper chromatography separate amino acids?

polarity

How well did you know this?

Not at all

Perfectly

what does paper chromatography separate?

amino acids

How well did you know this?

Not at all

Perfectly

what are the two solvent phases in amino acid chromatography? which one is mobile and which one is stationary? which substances are used in this experiment?

-non-polar mobile phase: isopropanol
-polar stationary phase: formic acid

How well did you know this?

Not at all

Perfectly

how does cellulose acetate electrophoresis separate proteins

by charge

How well did you know this?

Not at all

Perfectly

what is the formula for Rf?

distance moved by the sample from the line of origin/distance moved by the solvent from the line of origin

How well did you know this?

Not at all

Perfectly

how does cellulose acetate electrophoresis work?

Electrical current is passed over proteins moving through a porous cellulose acetate
membrane
(+) proteins move towards negative pole and (-) proteins move towards (+) pole

How well did you know this?

Not at all

Perfectly

what is serum (S)

Serum (S) is the blood plasma from which clotting factors (fibrinogen) have been removed. It contains electrolytes, hormones, proteins, and exogenous substances such as drugs. Its proteins can be separated into five protein fractions: albumin and alpha 1, alpha 2, beta and gamma globulins. Albumin is the main fraction, representing 60% of total proteins. Gamma globulins include immunoglobulins of which IgGs are the most abundant. Immunoglobulins, which are also known as antibodies (Figure 1), exhibit quaternary structures of two heavy chains and two light chains held by intermolecular disulfide bonds.

How well did you know this?

Not at all

Perfectly

what is fibrinogen?

clotting factors

How well did you know this?

Not at all

Perfectly

what are immunoglobulins?

Immunoglobulins, which are also known as antibodies (Figure 1), exhibit quaternary structures of two heavy chains and two light chains held by intermolecular disulfide bonds.

How well did you know this?

Not at all

Perfectly

what is the protein composition of serum S?

albumin and alpha 1, alpha 2, beta and gamma globulins. Albumin is the main fraction, representing 60% of total protein

How well did you know this?

Not at all

Perfectly

What is this

Immunoglobulin

How well did you know this?

Not at all

Perfectly

what are the two types of proteins in milk?

casein, a storage protein, makes up 80% of total proteins and whey proteins represent the remaining 20%

How well did you know this?

Not at all

Perfectly

what is the composition of egg whites?

90 percent water and 10 percent protein

How well did you know this?

Not at all

Perfectly

what type of protein do egg whites contain?

Up to 60% of total protein is made of ovalbumin, a high quality storage protein which is made of the most balanced combination of essential amino acids needed in human nutrition. Egg white also contains ovotransferrin, a glycoprotein, and a small amount of lysozyme which acts as an antimicrobial protease and digests bacterial cell walls.

How well did you know this?

Not at all

Perfectly

what does SDS stand for?

sodium dodecyl sulfate

how do we treat proteins before conducting SDS PAGE on them?

Before electrophoresis, proteins are treated with SDS (sodium dodecyl sulfate) and a reducing agent (DTT or BME) and boiled in order to denature them

what does SDS do to the proteins?

anionic detergent that binds to each protein evenly and imparts an even distribution of negative charge per unit mass

what does DTT and BME do?

used to disrupt disulfide bonds and to ensure complete denaturation and linearization of the proteins down to their primary structures

what do we stain the gel electrophoresis sample with?

Coomassie blue

by what property do we separate proteins with using SDS page?

by size

which size proteins travel the most with SDS page?

smaller ones

what is the isoelectric point?

PH at which the charge in a protein is 0

what does charge of protein depend on?

amino acid composition (the number of basic and acidic amino acidsin the R group) and on the pH of the solution in which it is dissolved

in SDS page, what pole to the proteins migrate towards?

positive (cause they're charged negatively)

what is the link between PI and PH?

At a pH higher than its pI a protein has a net negative charge (basic), provided that it consists of basic and acidic amino acids. Conversely, at a pH lower than its pI it has a net positive charge (acidic)

what is the support medium in cellulose acetate electrophoresis?

cellulose acetate

How are the protein patterns with cellulose acetate electrophoresis and SDS-PAGE different?

Cellulose Acetate Electrophoresis separates proteins by charge (negative or positive proteins move towards oppositely charged poles). SDS – PAGE separates proteins by size (molar mass) (small proteins migrate the most, large proteins migrate the least).

Explain why immunoglobulins appear as one fraction after electrophoresis on cellulose acetate but appear as heavy and light chains after SDS electrophoresis.

In SDS – PAGE, proteins are denatured in the presence of the anionic detergent SDS and the reducing agent beta-mercaptoethanol. Therefore, the heavy and light chains are separated and migrate as individual polypeptides through the SDS – polyacrylamide gel. In cellulose acetate electrophoresis, proteins retain their tertiary and quaternary structures.

Why do you not see lysozyme on the SDS polyacrylamide gel?

Lysosyme has a MM of 14.3 kDa. Being small, it has migrated out of the 10% polyacrylamide gel and does not appear on it. However, lysozyme is visible on the 12% polyacrylamide gel.

What is the charge of ovalbumin and lysozyme?

Ovalbumin: negative Lysozyme: positive

why does casein not dissolve in water?

Purified casein does not readily dissolve in water as it is highly hydrophobic

what does casein form in milk?

In milk, it forms casein micelles, complex quaternary structures in which individual casein polypeptides are held together by hydrophobic interactions and by chelating calcium and phosphate ions

when do casein proteins coagulate?

in the process of cheese making

what is the point of the amino acid chromatography experiment? what is our control?

the point is the identify which amino acids are present in an unknown sample of protein, using the rf values of our known amino acids

why does casein not migrate in cellulose acetate electrophoresis?

does not migrate due to its size and hydrophobic nature inside the micelle

which one is too small to visualize?

lysosome

what does ponceau s do?

stains cellulose acetate bands to better visualize them