Transcript Bradford Assay

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?
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?
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)
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
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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
𝑚𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛
𝑚𝑙
12 𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 1000𝑚𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 50.𝑚 𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛
(
)(
)=
240 𝑚𝑙
𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛
𝑚𝑙
• 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?
•
50.𝑚 𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 1
( )
𝑚𝑙
125
=
0.40 𝑚 𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛
𝑚𝑙
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?
Absorbance (+/-0.001) at 864 nm
Determining concentration of 1/125 mutant milk dilution
0.500
0.450
0.400
0.350
0.300
0.250
0.200
0.150
0.100
0.050
0.000
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Concentration of Bovine gamma Globulin standard y = 0.6114x
(mg protein/ml)
R² = 0.9136
average absorbance
Linear (average absorbance)
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?
Absorbance (+/-0.001) at 864 nm
Determining concentration of 1/125 mutant milk dillution
0.500
0.450
0.400
0.350
0.300
0.250
0.200
0.150
0.100
0.050
0.000
0
0.1
0.2
0.3
0.4
0.5
0.6
Concentration of Bovine gamma Globulin standard
(mg protein/ml)
average absorbance
Linear (average absorbance)
0.7
0.8
y = 0.6114x
R² = 0.9136
Protein Concentration: silly example
What does the R2 value tell you?
Determining concentration of 1/50 mutant milk dillution
0.500
0.450
absorbance at 864 nm
•
0.400
0.350
0.300
0.250
0.200
0.150
0.100
0.050
0.000
0
0.1
0.2
0.3
0.4
0.5
0.6
Concentration of Bovine gamma Globulin standard
(mg protein/ml)
average absorbance
Linear (average absorbance)
0.7
0.8
y = 0.6114x
R² = 0.9136
Protein Concentration: silly example
How can you test your accuracy?
% 𝑒𝑟𝑟𝑜𝑟 =
𝑜𝑏𝑠𝑒𝑟𝑣𝑒𝑑 −𝑒𝑥𝑝𝑒𝑐𝑡𝑒𝑑
𝑒𝑥𝑝𝑒𝑐𝑡𝑒𝑑
x100%
Determining concentration of 1/50 mutant milk dillution
0.500
0.450
absorbance at 864 nm
•
0.400
0.350
0.300
0.250
0.200
0.150
0.100
0.050
0.000
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Concentration of Bovine gamma Globulin standard y = 0.6114x
(mg protein/ml)
R² = 0.9136
average absorbance
Linear (average absorbance)