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Bradford Assay How much protein is really in my… -milk? -soy “milk”?

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Presentation on theme: "Bradford Assay How much protein is really in my… -milk? -soy “milk”?"— Presentation transcript:

1 Bradford Assay How much protein is really in my… -milk? -soy “milk”?

2 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

3 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?

4 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)?

5 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

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

7 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

8 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)

9 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)

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

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

12 Protein Concentration: silly example

13 -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?

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


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