How much protein is really in my… -milk? -soy “milk”? 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! 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 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 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 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 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? How could you evaluate the accuracy of the lab results?...percent error [observed-expected]/expected X100% How could you evaluate the precision of the lab results?...trend lines R2 value, error bars

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 𝑚𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑚𝑙 ( 12 𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 240 𝑚𝑙 )( 1000𝑚𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 )= 5.0𝑚 𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑚𝑙 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? 5.0𝑚 𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑚𝑙 ( 1 50 ) = 0.10 𝑚 𝑔 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑚𝑙

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? 0.275=0.6114X X= 0.45 mg protein/ml Percent error: .45-.50/.50X100=10%

Protein Concentration: silly example What does the R2 value tell you? Precision! How well does the trend line equation predict the data points 0.95 ok for “student” research, industry 0.98….some even require 0.99- think about how important it is that pharmaceuticals are made precisely the same predictable way over and over again! The only way you get a R2=1 is if you have 2 data points or you do your research in Eutopia