QUIZ TIME! Please hand in your Lab 5 Report You have 5 minutes (from 2:10 to 2:15 pm) to complete the quiz 1. Silence and put away your cell phone/digital devices 2. Have your calculator available in case you may need it for the quiz 3. Work on your own; no cheating 4. When done, turn your quiz upside down and raise your hand once for a TA or instructor to come and collect your quiz 5. Please keep quiet and do not open your lab manual or lecture related notes until all quizzes have been collected. Lab 2: Quantification of Protein - Bradford Assay • Qualitative v. Quantitative • Quantification: determining how much protein is in a sample (compare to known; STD CURVE) • Bradford Assay: an accurate and sensitive method for determining the concentration of proteins (peptide bonds) ₋ Can detect as little as 1 μg/mL of protein ₋ Utilizes Brilliant blue dye that changes color in the presence of proteins No Protein Protein Bradford Assay: Colorimetric Method + Protein • Color change allows you to determine the amount of protein: ₋ The more protein there is the darker the blue color will be Bradford Assay: Spectrophotometer ₋Intensity of the blue color can be measured using a spectrophotometer (converts color intensity to numbers) ₋The higher the protein concentration, the darker the color, the higher the abs. # Bradford Assay: Maximal Absorbance Change at 595 nm Protein + Dye [BSA] µg/ml 2000 1500 1000 750 500 250 125 0 www.thermofisher.co m – Protein + Protein Bradford Assay: Known Amount of Proteins as Standards 0 μg/mL 400 μg/mL • We can measure known protein concentrations to generate a standard curve • We can then plot the unknowns to the curve to determine their concentration Today’s Lab • Turn spec on Preparing different concentrations (STD curve) from Stock C1V1 = C2V2 Prepare 10 ml of 2 M NaCl using 5 M stock 5X= 2 (10) X = 4 ml In a flask/tube add 6 ml of H2O to 4 ml of NaCl stock = 10 ml of 2 M NaCl * Using BSA stock solution (1 mg/ml) prepare 1000 l of 50, 100, 200, 300 and 400 g/ml Amount of BSA Stock (1 mg/ml) Amount of Water • Dilution factor = Final volume/sample volume Final [BSA] (g/ml) 50 100 200 300 400 Today’s Lab Obtain 15 microfuge tubes. Mark one tube Blank add 50 l H2O 2 tubes unknown 1 add 50 l to each tube 2 tubes unknown 2 add 50 l to each tube 2 tubes for each standard (total 10 tubes for standards), add 50 l unknown to each tube • Add 1500 l Bradford Reagent to each tube • Wait 5 min 1 blank (water) 4 unknown (sample) sample • • • • • Prepare 5 dilutions of 1 mg/mL BSA (duplicates) Add 1.5 mL of Bradford reagent to all tubes Today’s Lab Stock 1mg/ml BSA A B + + + + + C ADD 950 900 800 700 600 l H2O D Transfer 50 l 50 l 50 l 50 l 50 l STD Conc. F E • • • • • Turn spec on Obtain 20 microfuge tubes. Mark 3 tubes for each standard one tube Blank 2 tubes unknown 1 2 tubes unknown 2 Blank/H2O 50 l Unknown 1 Unknown 2 50 l 50 l 100 200 50 300 400 g/ml g/ml g/ml g/ml g/ml Add 1500 l Bradford/tube (all 15 tubes) Incubate Use 1 ml to measure absorbance SAVE B, STD & SAMPLES Today’s Lab • Transfer 1.5 ml to cuvette to measure abs. • Rinse cuvette, wipe finger prints, avoid air bubbles • Blank Spectrophotometer using the sample prepared with water • Measure absorbance at 595 nm (going from the lowest → highest concentration) • Rinse and reuse microcuvettes • Record absorbance and average your duplicates • Graph absorbance (Y-axis) versus protein concentration (X-axis) • Use fitted graph to find concentrations of the 2 unknowns Standard Curve to Determine Concentration of Unknowns Bradford Assay Standard Curve Absorbance (595 nm) 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 0 20 10 20 30 30 40 50 40 50 60 70 Concentration (μg/ml) 80 60 90 • Graph Absorbance (595 nm; y-axis) versus BSA concentration (µg/ml; xaxis) to obtain your standard curve • Measure Absorbance (595 nm) of your unknowns and use the standard curve to determine their protein concentration (e.g., unknown Abs of 0.25 corresponds to 54 µg/ml protein concentration [follow the red arrows]) • Wear gloves when handling BSA, protein, and the cuvettes (fingerprints on the cuvettes and bubbles in your samples in the cuvette can affect the absorbance readings) • After you have measured and recorded the absorbance of your samples, you should wash your protein solutions down the sink • Microfuge tubes and pipette tips can go into the regular trash • Check out with the TAs when you have completed the lab Biology 115 Lab Fall 2018 Lab 2 Worksheet Instructor: _____________________________________________________________________________________ I. Student Name: Group Members: _____________________________________________________________________________________ II. Personalized Title [0.5 pts] _____________________________________________________________________________________ III. Hypothesis [1.0 pts] 1. _____________________________________________________________________________________ IV. Introduction IVA. General Theory/Background [1.0 pts] IVB. Summary of Overall Approach [1.5 pts] Page 1 of 4 Biology 115 Lab Fall 2018 Lab 2 Worksheet Instructor: _____________________________________________________________________________________ V. Observations & Results VA. Data [1.5 pts] Page 2 of 4 Biology 115 Lab Fall 2018 Lab 2 Worksheet VB. Calculations Instructor: [0.5 pts] VI. Discussion Questions 1. What If you would forget to use blank and calculated your unknown sample protein concentration using standards. [0.5 pt] 1. What would you do if your unknown sample had an A595 greater than that of your BSA standards? What if the unknown A595 was less than your BSA standards? [0.5 pt] 2. Why is 595 nm used in Bradford assay? What about 450 nm? [0.5 pt] Page 3 of 4 Biology 115 Lab Fall 2018 Lab 2 Worksheet Instructor: _____________________________________________________________________________________ VII. Conclusion(s)/Inference(s): [0.5 pt] _____________________________________________________________________________________ Total Score: _____ / 8.00 (Includes _____ Bonus Points for ________________________________________________) Comments: ___________________________________________________________________________ _____________________________________________________________________________________ Page 4 of 4 
Purchase answer to see full attachment