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 = h

h = 6.626 X 10-34 J s

= 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

c =

Replacing in E equation

E = hc/

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

C = concentration of EMR-absorbing substances

= extinction coefficient (absorptivity)

Relates the amount of light absorbed to the concentration

L = path length

Proteins Absorb at 280 nm 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

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

Very time and temperature dependent reactions!

Dye-Binding (Bradford) Assay

A blue dye is used

Coomassie Brilliant Blue G250

Absorbs light at 465 nm (max)

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

g/mL Protein

. .

. .

.

Standard Curve

A

g/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 g/mL

Requires 10 L of Stock and 990 L buffer

Dilute this Second Stock to 100 ng/mL

Requires 10 L of New Stock and 990 L buffer

Dilute Third stock 10-fold to 10 ng/mL

Requires 100 L Third Stock with 900 L buffer

When May a Serial Dilution be Necessary?

A

g/mL Protein

. .

. .

.

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

Micropipets Use to dispense liquids > 1 mL

P1000 : 200 l 1000 l

P200: 20 l 200 l

P20: 2 l 20 l

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