Biochemistry I Discussion and Lab BI/CH 421/621/527

Transcription

1 Biochemistry I Discussion and Lab BI/CH 421/621/527 Fall 2012 TF: Sarah Soltau, ssoltau@buedu Office Hours: M 12-1 pm & Tu 2:30-3:30 pm, SCI 161 Discussions: M am, KCB 106 Tu 11 am -12 noon, SAR 300 W am, SAR 300 Labs (SCI 162): W 8 am 12 noon, 1 5 pm, 6 10 pm Th 8 am 12 noon, 1 5 pm, 6 10 pm F 10 am 2 pm, 3 7 pm M 11 am -3 pm, 4 8 pm

2 Biochemistry Discussion Goal to provide information necessary to understand the lab and complete it efficiently Quiz at end of each discussion Lab Exam during last discussion section Lab Grading: Item Weight Discussion Quizzes 20% Lab Write-Ups 60% Comportment 10% Lab Exam 10%

3 Biochemistry Lab Guidelines Need lab manual, safety glasses, and bound laboratory notebook Must wear closed-toed shoes and long pants, lab coats encouraged Read lab manual thoroughly BEFORE lab Write your own procedure into your lab notebook before coming lab lab manuals will not be permitted in the lab Try to meet with lab partner before lab to plan out experiments ahead of time to finish on time

4 Biochemistry Lab Guidelines Problems in each chapter are optional Help to guide post-lab analysis in notebook section Answers are in appendix of each chapter Write down all data in Notebook section during lab Answer all calculations and questions in Notebook section Show all sample calculations

5 Biochemistry Lab Guidelines Can discuss with post-lab with your lab partner, but work must be done separately Academic integrity policy Any plagiarism is a violation and results in a zero on the assignment Plagiarism will be referred to the to Dean of Academic Affairs Labs due one week after completion in the lab late penalty of 20% per day, weekends count as 2 days Labs will returned within 48 hours to boxes outside the lab, SCI 162

6 Laboratory Notebook Format Introduction Experimental Procedure Notebook Section Experimental Data Calculations Graphs, Pictures, Other results Answers to questions (NOT Problems, these are for your own benefit, answers are in the appendix) Discussions/Conclusions No Pencil or White-Out in Lab Notebooks!

7 Chapter 1: Photometric Methods for Protein Determination Purpose: To become familiar with three widely-used methods of determining the concentration of proteins in a solution Methods: Lowry Dye-binding Direct UV absorbtion

8 Review of Spectroscopy Electromagnetic radiation (EMR) ranges from high energy (cosmic rays) to low energy (radiowaves) E = hν h = 6626 X J s ν = frequency of EMR The greater the frequency the greater the E Due to electronic configuration functional groups absorb EMR at different frequencies

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10 Laboratory Measurements Use Wavelength Spectrophotometers UV/Vis measures Absorbance at λ c = λ ν Replacing ν in E equation E = hc/λ

11 Absorbance Units & Beer's Law The more EMR-absorbing substances present in a sample yields less light transmitting through the sample This can give us a quantitative measurement of the EMR-absorbing substances Beer's Law: A = -log T = c L ε C = concentration of EMR-absorbing substances ε = extinction coefficient (absorptivity) Relates the amount of light absorbed to the concentration L = path length

12 Proteins Absorb at 280 nm λ max in proteins is 280 nm due to Trp and Tyr The more Trp and Tyr, the greater the A of EMR However, different proteins contain different amounts of Tyr and Trp Direct UV absorption data cannot tell you [Protein] without ε

13 Lowry Method of Protein Determination Method is a combination of reactions Biuret Reagent Alkaline solution of copper ions Turn pink/purple when exposed to protein backbone Folin-Ciocalteu reagent - Cupric Reagent (blue) Dependent upon reducing power of aromatic amino acids so not very dependable on its own Also dependent upon the number of aromatic residues This method is more dependable than UV but suffers from protein-to protein variation of ε Very time and temperature dependent reactions!

14 Dye-Binding (Bradford) Assay A blue dye is used Coomassie Brilliant Blue G250 Absorbs light at 465 nm (λ max ) Upon binding protein absorbs light at 595 nm Small effects of buffer Blanking the dye with buffer compensates

15 Helps quantify [Protein] Standard Curves Lowry curve will vary from protein to protein Bradford curve should be the same from protein to protein Standard Curve To measure [Protein] Find A A A µg/ml Protein µg/ml Protein

16 Observing Interference While Quantifying [Protein] To observe and quantify interference you should Get actual [Protein] without IS Run assay with IS without protein Run assay with IS and protein This will give you correction values to compensate for IS (p 33)

17 Serial Dilutions Sometimes serial dilutions are needed to dilute a substance to a workable concentration Standard Dilution M 1 V 1 = M 2 V 2 To make 100 ml of a 010 mg/ml solution from a 10 mg/ml stock Use 0100 ml of Stock and 0900 ml appropriate buffer 0100 ml is an easily-measured volume

18 Serial Dilutions To make a 10 ng/ml solution from the same stock Need ml of Stock not easily measured Serial dilution necessary Dilute from 10 mg/ml to 10 µg/ml Requires 10 µl of Stock and 990 µl buffer Dilute this Second Stock to 100 ng/ml Requires 10 µl of New Stock and 990 µl buffer Dilute Third stock 10-fold to 10 ng/ml Requires 100 µl Third Stock with 900 µl buffer

19 When May a Serial Dilution be Necessary? Your solution's A reading Is MUCH higher than the Standard Curve A µg/ml Protein

20 Micropipets Use to dispense liquids > 1 ml P1000 : 200 µl 1000 μl P200: 20 µl 200 µl P20: 2 µl 20 µl Push plunger to first stop to draw in liquid Release plunger slowly Push plunger to the second stop to release all liquid in the tip

21 What Should Be In Your Notebook Before Lab?

22 What Should Be In Your Notebook Before Lab? Introduction 3-4 sentences, goals of experiment Procedure In your own words, not copied from lab manual Setup/Materials Data tables and space to write down experimental results during lab