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Bradford Assay

Bradford Assay

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Bradford Assay

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  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

  3. Protein Qualitative v. Quantitative • But what if you want to know the concentration of protein in a solution?...how can this be quantified

  4. Where do these numbers come from? Both Soy “milk” and Cow milk have the same serving size: 1 cup…AKA 240ml

  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 Smart Spec Plus by BioRad • 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”

  8. How do we measure protein concentration?Activity 7.1 Youtube: Bradford Assay by BioRadLifeSciencehttp://www.cleanvideosearch.com/media/action/yt/watch?v=vfY3mVOlGBU&sns=em Spectrophotometer Smart Spec Plus by BioRad • Before determining unknown protein concentration (Unit read out will be in mg/ml) • Generate a standard curve of known protein concentrations! • SmartSec can save a standard curve…modify Activity 7.1 accordingly • you will only need to make unknown samples • Also, Ms. McKay will make a blank that everyone can share

  9. Protein Concentration • Use Milk nutritional information to determine expected protein. • In a 1cup serving size (240ml) there are 8g protein • Give answer in • Wait! Don’t we dilute the milk before we measure the protein concentration in the SmartSpec? • By what factor will the milk be diluted? 1/50 • So in an ideal world, with an ideal cow, and “perfect” lab technique, how many mg or protein do you expect to measure per ml of 1/50th diluted milk?

  10. Prelab • Review p220-221 • Activity 7.1 • Read over Overview, tips and notes…etc • Can you answer Prelab Focus Questions? • In lab notebook • Purpose:…. • Procedure: note that steps 6-8 are predone! Skip step 10! Note step 11 predone also!!! Stop after step 13!