Bradford Assay

How much protein is really in my…-milk?

-soy “milk”?-almond “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!Blue- no protein

Purple – yes! protein

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?

7 minutes- in lab groups-use internet access tool to find out either

1) Nutritional information about a brand of almond milk, soy milk, or cows milk.- record

the source!2) How much protein in a serving?

3) How big is a serving in milliliters?4) What is the concentration of protein

(mg/ml)?

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

Which tube has a greater protein concentration…3 or 7?1 or 8?

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 color?

• Measures a specific wavelength of light absorbed by material.• For measuring Bradford reacting w/ protein- use 595nm wavelength

Spectrophotometer (published absorbance uncertainty is +/- 0.001)

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

filterCuvette (sample) Detector

Data Display

(absorbance)

How do we measure protein concentration?FYI:Youtube: Bradford Assay by BioRadLifeScience

http://www.cleanvideosearch.com/media/action/yt/watch?v=vfY3mVOlGBU&sns=em uses 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?

How do we measure protein concentration?FYI:Youtube: Bradford Assay by BioRadLifeScience

http://www.cleanvideosearch.com/media/action/yt/watch?v=vfY3mVOlGBU&sns=em uses 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• 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

1/125• 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 1/125th diluted milk?

• () =

Protein Concentration: silly example• -lets say this mutant milk reacted w/ PseudoBradford, but it required absorbance

readings at 864nm. We tested various protein concentration to create a standard curve.

• What does this graph mean?

0.2 0.3 0.4 0.5 0.6 0.7 0.80.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.400

0.450

0.500

f(x) = 0.611428571428571 xR² = 0.992403190703245

Determining concentration of 1/125 mutant milk dilution

average absorbance Linear (average absorbance)

Concentration of Bovine gamma Globulin standard (mg protein/ml)

Abso

rban

ce (+

/-0.

001)

at 8

64 n

m

Protein Concentration: silly example• The average absorbance (at 864nm) taken from the spectrophotometer was 0.275

+/-0.001. Using thin information and the graph, what would be the observed protein concentration of the diluted milk?

0.2 0.3 0.4 0.5 0.6 0.7 0.80.0000.0500.1000.1500.2000.2500.3000.3500.4000.4500.500

f(x) = 0.611428571428571 xR² = 0.992403190703245

Determining concentration of 1/125 mutant milk dillution

average absorbance Linear (average absorbance)

Concentration of Bovine gamma Globulin standard (mg protein/ml)

Abso

rban

ce (+

/-0.

001)

at 8

64 n

m

Protein Concentration: silly example• What does the R2 value tell you?

0.2 0.3 0.4 0.5 0.6 0.7 0.80.0000.0500.1000.1500.2000.2500.3000.3500.4000.4500.500

f(x) = 0.611428571428571 xR² = 0.992403190703245

Determining concentration of 1/50 mutant milk dillution

average absorbance Linear (average absorbance)

Concentration of Bovine gamma Globulin standard (mg protein/ml)

abso

rban

ce a

t 864

nm

Protein Concentration: silly example• How can you test your accuracy?

x100%

0.2 0.3 0.4 0.5 0.6 0.7 0.80.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.400

0.450

0.500

f(x) = 0.611428571428571 xR² = 0.992403190703245

Determining concentration of 1/50 mutant milk dillution

average absorbance Linear (average absorbance)

Concentration of Bovine gamma Globulin standard (mg protein/ml)

abso

rban

ce a

t 864

nm