Protein & Protein Quantification (Bradford Assay)

RECAP

  • one gene, one protein - cells produce many different proteins hence every different protein is encoded with a different gene

  • stop codons - UAA, UGA, UAG

  • start codon - AUG

  • carboxyl group of one amino acid is linked to the amino group of another amino acid - known as dehydration reaction and a peptide bond is formed

  • (levels of folding) primary - secondary - tertiary - quaternary

  • peptide bonds are not broken during denaturation and only unfold into a random coil

Bradford Assay

purpose: to measure the amount of total protein in the broth

pros and cons

pros

  • fast, inexpensive and sensitive

  • tolerates a wide range of buffers

cons

  • protein dependant (react primarily with arginine)

  • incompatible with many detergents

  • coomassie dye binds to quartz so it is advisable to use glass or plastic cuvettes

  • (basically the proteins it can measure are restrictive so for wider range of different proteins use the other method)

principles

  • coomassie blue dye has max absorbance at 465nm

  • when the dye binds to protein molecules (esp arginine), max absorbance changes to 595nm

how to perform the assay

  1. dissolve 100mg coomassie brilliant blue G-250 in 50ml, 95% ethanol, add 100ml 85% (w/v) phosphoric acid. Dilute to 1 liter when the dye has completely dissolved and filter through Whatman #1 paper just before use

  2. dilute unknown protein sample to obtain between 5 and 100µg protein (have 8 different tubes) in 100µl volume

  3. add 5 ml dye reagent and incubate 5 min

  4. aliquot each protein solution into a cuvette and slot it into a spectrophotometer

  5. measure the absorbance at 595nm

  6. plot graph using OD readings (bradford assay graphs requirements below)

    1. pass through (0,0)

    2. best fit straight line

    3. ignore outliers at end point

spectrophotometric assay at 280nm

purpose: to measure the amount of total protein in the broth

pros and cons

pros

  • fast and convenient, since no additional reagents or incubations are required

  • no protein standard need be prepared

cons

  • Tryptophan absorbs 280nm the highest, so the absorbance of a protein is very heavily influenced by the Tryptophan content (false readings can happen very often)

    • proteins contain little or none of tryptophan have very low absorbance (false low)

    • proteins that have unusually high amounts of this amino acid will absorb too strongly (false high)

  • can only use quartz cuvette - because plastic absorbs UV

principle

  • amino acids containing aromatic rings such as trytophan, tyrosine, histidine and phenylalanine residues absorb a great amount of light at UV range (280nm)

  • simple terms: amino acids will absorb a lot of light so different light absorbance and use formula to figure out the protein conc.

calc of protein conc

  • Conc (mg/ml) = 1.55 x distance travelled at 280nm