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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. Protein Qualitative v. Quantitative. - PowerPoint PPT Presentation
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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
Protein Qualitative v. Quantitative
• But what if you want to know the concentration of protein in a solution?...how can this be quantified
Where do these numbers come from?
Both Soy “milk” and Cow milk have the
same serving size: 1 cup…AKA 240ml
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– 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”
Smart Spec Plus by BioRad
How do we measure protein concentration?Activity 7.1
Youtube: Bradford Assay by BioRadLifeScience http://www.cleanvideosearch.com/media/action/yt/watch?v=vfY3mVOlGBU&sns=em
Spectrophotometer– 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
Smart Spec Plus by BioRad
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?
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!