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

Discussions: M 10-11 am, KCB 106 Tu 11 am -12 noon, SAR 300 W 10-11 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

Fall 2012

TF: Sarah Soltau, ssoltau@bu.edu

Office Hours: M 12-1 pm & Tu 2:30-3:30 pm, SCI 161

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%

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

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

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

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!

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

Review of Spectroscopy

● Electromagnetic radiation (EMR) ranges from high energy (cosmic rays) to low energy (radiowaves)

● E = hν

● h = 6.626 X 10-34 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

Laboratory Measurements Use Wavelength

● Spectrophotometers

● UV/Vis measures Absorbance at λ

● c = λ ν

● Replacing ν in E equation

E = hc/λ

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

Proteins Absorb at 280 nm ● λmax 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

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!

Dye-Binding (Bradford) Assay

● A blue dye is used

● Coomassie Brilliant Blue G250

● Absorbs light at 465 nm (λmax)

● Upon binding protein absorbs light at 595 nm

● Small effects of buffer

– Blanking the dye with buffer compensates

Standard Curves ● Helps quantify [Protein]

● Lowry curve will vary from protein to protein

● Bradford curve should be the same from protein to protein

A

µg/mL Protein

. .

. .

.

Standard Curve

A

µg/mL Protein

. .

. .

.

To measure [Protein] Find A

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)

Serial Dilutions

● Sometimes serial dilutions are needed to dilute a substance to a “workable” concentration

● Standard Dilution

M1V1 = M2V2

● To make 1.00 mL of a 0.10 mg/mL solution from a 1.0 mg/mL stock

● Use 0.100 mL of Stock and 0.900 mL appropriate buffer

● 0.100 mL is an easily-measured volume

Serial Dilutions ● To make a 10 ng/mL solution from the same stock

● Need 0.0000100 mL of Stock – not easily measured

● Serial dilution necessary

● Dilute from 1.0 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

When May a Serial Dilution be Necessary?

A

µg/mL Protein

. .

. .

.

Your solution's A reading Is MUCH higher than the Standard Curve

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

What Should Be In Your Notebook Before Lab?

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