Protein isolation module

       What molecules make up proteins and how are they joined together?

Amino Acids, peptide bonds

     

What part of an amino acid defines its chemical properties?

R group, side chain

     

Define the 4 levels of protein structure.

Primary is a linear segment of amino acids,

Secondary is primary with it meeting up with it s self, from hydrogen bonds Beta sheets alpha helixes,

Tertiary is folded due to R-group interaction which has function.

Quatranary is multipile tertiary proteins interacting with each other to form a function.

      What is denaturation and what environmental changes can cause it?

The unlfolding of a protien that chnages function,

• heat,

• ph change,

• ion concentration,

• detergents like SDS,

• organic solvents,

• mechanical stress

      How is a protein concentration standard curve created?

Prepare known concentrations of a standard protein (usually BSA), measure absorbance,

plot absorbance vs concentration,

and fit a line.

      What is the purpose of BPER?

BPER = Bacterial Protein Extraction Reagent → chemically lyses cells to release proteins.

 What is the relationship between the Bradford Coomassie Assay, protein concentration and spectrophotometry?

Coomassie dye binds proteins and turns blue. The amount of color (absorbance at 595 nm) is proportional to protein concentration → measured with a spectrophotometer.

 If you use the assay mentioned above and measure an absorbance outside of your standard curve, can you accurately determine the protein concentration? How could you fix this problem?

No, you can’t accurately determine it. Dilute your sample until it falls inside the curve, then multiply back.

 What is the purpose of adding SDS to your protein sample prior to running it on an SDS PAGE?

SDS denatures proteins and gives them uniform negative charge proportional to length → separates by size only.

 What is the premise of SDS-PAGE?

How did we visualize our proteins?

What can we determine from the gels?

How will denatured / non-denatured samples differ from each other?

What direction do samples run (positive or negative)?

We use uv lights,

we can determine the amino acid length, and potential protein identity,

they run towards the postive because the proteins are negative

 Know how to use an experimental absorbance value and standard curve to determine protein concentration, know how to prepare a 10ug SDS-PAGE sample from this value.

C=M/V concentration is direct to absorbance therefor mass too.