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Bradford Assay. How much protein is really in my… -milk? -soy “milk”?. Protein Qualitative v. Quantitative. Qualitative: Which of the following chemicals turns purple in the presence of protein? Lugols iodine Benedicts solution Biuret Solution. Biuret tests for protein !

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bradford assay

Bradford Assay

How much protein is really in my…

-milk?

-soy “milk”?

protein qualitative v quantitative
Protein Qualitative v. Quantitative
  • Qualitative: Which of the following chemicals turns purple in the presence of protein?
    • Lugols iodine
    • Benedicts solution
    • Biuret Solution

Biuret tests for protein!

Blue- no protein

Purple – yes! protein

protein qualitative v quantitative1
Protein Qualitative v. Quantitative
  • But what if you want to know the concentration of protein in a solution?...how can this be quantified…and why would we care?
where do these numbers come from
Where do these numbers come from?

7 minutes- in lab groups

-use internet access tool to find out either

Nutritional information about a brand of almond milk, soy milk, or cows milk.- record the source!

How much protein in a serving?

How big is a serving in milliliters?

What is the concentration of protein (mg/ml)?

protein quantitation p220 221
Protein Quantitation p220-221
  • Bradford Assay-developed 1976
    • Coomassie Brilliant Blue G-250 (AKA Bradford reagent) interacts w/ specific amino acids (especially the R-group on the amino acid arginine)
      • Protein Absent: redish-Brown
      • Protein present: blue (peak absorbance between 470nm & 595nm)
        • Amount of blue is proportional to protein concentration
          • The greater the protein concentration the more intense the blue color is!

Semi Micro Cuvettes

how do we measure color
How do we measure color?

Spectrophotometer (published absorbance uncertainty is +/- 0.001)

  • Measures a specific wavelength of light absorbed by material.
  • For measuring Bradford reacting w/ protein- use 595nm wavelength
how do we measure color1
How do we measure color?

Spectrophotometer (published absorbance uncertainty is +/- 0.001)

  • Measures a specific wavelength of light absorbed by material.
  • For measuring Bradford reacting w/ protein- use 595nm wavelength
how do we measure color2
How do we measure color?

Spectrophotometer

  • Measures a specific wavelength of light absorbed by material.
    • Narrow beam of light at set wavelength shines through cuvette and sample…how much light makes through to the other side to be detected by the “detector”

Light source

prisim

Wavelength filter

Cuvette

(sample)

Detector

Data Display

(absorbance)

slide10

How do we measure protein concentration?FYI:Youtube: Bradford Assay by BioRadLifeSciencehttp://www.cleanvideosearch.com/media/action/yt/watch?v=vfY3mVOlGBU&sns=emuses more “biotech” equipment Smart Spec Plus that can retain a standard curve

Experiment part 1

  • Make a “blank” cuvette – using 50µl 1XPBS (unreactive filler chemical) + 2.5ml of Bradford reagent
    • What would you use the “blank” cuvette for?

Experiment part 2

  • Before determining unknown protein concentration: Use at least 4 protein samples that have a known protein concentration – ex: Bovine Gamma Globulin (BGG)…using 50µl of given [BGG]+2.5ml of Bradford reagent- and determine their absorbance at 595nm wavelegth
      • Why do you need this?
      • What would be the IB minimum number of readings to take from each sample?
slide11

How do we measure protein concentration?FYI:Youtube: Bradford Assay by BioRadLifeSciencehttp://www.cleanvideosearch.com/media/action/yt/watch?v=vfY3mVOlGBU&sns=emuses more “biotech” equipment Smart Spec Plus that can retain a standard curve

Experiment part 3

  • Determine the absorbance (at 595nm wavelength) of 50µl diluted milk mixed with 2.5 ml Bradford reagent
    • Once you process your data, what should you be able to determine?
    • Why do you think we must use diluted milk in this experiment?
    • Could these results be compared against published results?
    • How could you evaluate the accuracy of the lab results?
    • How could you evaluate the precision of the lab results?
protein concentration silly example
Protein Concentration: silly example
  • Use Mutant Milk nutritional information to determine expected protein.
    • In a 1cup serving size (240ml) there are 12g protein
      • Give answer in
      • ()()=
    • Wait! Don’t we dilute the milk ?
    • By what factor will the milk be diluted? Lets say it was

1/50

      • So in an ideal world, with an ideal cow, and “perfect” lab technique, how many mg or protein do you expect to per ml of 1/50th diluted milk?
      • () =
protein concentration silly example1
Protein Concentration: silly example
  • -lets say this mutant milk reacted w/ PseudoBradford, but it required absorbance readings at 864nm
  • If the average absorbance (at 864nm) was 0.275 +/-0.001, then what would be the observed protein concentration of the diluted milk? What would be the percent error?
protein concentration silly example2
Protein Concentration: silly example
  • What does the R2 value tell you?