96

Lab 11: Studying Protein Concentration; GFP Purification, Part 1 Today you will begin the process of purifying the GFP protein from the bacterial cells that you

transformed with the pGLO plasmid two weeks ago. You will continue the GFP purification procedure next week in Lab 12. Today’s lab will also teach you how to use the spectrophotometer to determine the concentration of a protein solution using the Bradford assay. You will use this assay again later to determine the concentration of your purified GFP protein.

Activity 11a GFP Purification, Part 1 Purpose

The overall purpose of the GFP purification procedure is to isolate (purify) the GFP protein from the mixture of bacterial cellular proteins in the pGLO-transformed E. coli cells. In today’s activity, you will begin the purification process by harvesting the E. coli cells and lysing them (breaking them open to release their cellular contents).

Before beginning the laboratory activities, we will first learn more background on how the GFP

purification procedure works in a Powerpoint lecture (see next 3 pages for notes).

97

GF

P P

urific

atio

n

GF

P

Pu

rifica

tion

Pro

ce

du

re,

GF

P

Pu

rifica

tion

Pro

ce

du

re,

(co

nt.)

Why U

se

Chro

mato

gra

phy?

!!T

o p

urify

a s

ingle

recom

bin

ant p

rote

in

of in

tere

st fro

m o

ver

4,0

00 n

atu

rally

occurrin

g E

. coli

gene p

roducts

.

98

Colu

mn

Chro

mato

gra

phy

!!C

hro

mato

gra

phy

used fo

r pro

tein

purific

atio

n

"!S

ize e

xclu

sio

n

"!Io

n e

xch

an

ge

"!H

yd

rop

ho

bic

inte

ractio

n

Hydro

phobic

In

tera

ctio

ns

In h

igh

sa

lt, hyd

rop

ho

bic

p

arts

of p

rote

ins b

eco

me

e

xp

ose

d o

n th

e s

urfa

ce

o

f the

pro

tein

Hig

h s

alt b

uffe

r fo

r bin

din

g

Th

is re

su

lts in

the

hyd

rop

ho

bic

pro

tein

s s

tickin

g

tog

eth

er m

ore

b/c

hyd

rop

ho

bic

mo

lecu

les lik

e

oth

er h

yd

rop

ho

bic

mo

lecu

les

Fo

r the

sa

me

rea

so

n, h

yd

rop

ho

bic

pro

tein

s w

ill als

o s

tick

to a

hyd

rop

ho

bic

ch

rom

ato

gra

ph

y c

olu

mn

Hydro

phobic

In

tera

ctio

ns

Hy

dro

ph

ob

ic

be

ad

s

Sin

ce

GF

P is

a v

ery

h

yd

rop

ho

bic

pro

tein

, it w

ill bin

d to

the

h

yd

rop

ho

bic

be

ad

s o

n

the

co

lum

n m

atrix

in th

e

pre

se

nce

of h

igh

sa

lt …

An

d w

ill co

me

off th

e

co

lum

n (e

lute

) with

a

ve

ry lo

w s

alt b

uffe

r . . .

. . . be

ca

use

w/o

hig

h s

alt, th

e h

yd

rop

ho

bic

pa

rts o

f the

pro

tein

will b

e b

urie

d in

in th

e c

ore

of th

e p

rote

in a

ga

in, s

o th

e p

rote

in w

ill no

lon

ge

r bin

d to

the

co

lum

n.

Hydro

phobic

Inte

ractio

n

Chro

mato

gra

phy (H

IC):

Ste

ps 1

–3

1.!

BIN

D: A

dd

ba

cte

rial ly

sa

te to

co

lum

n m

atrix

in h

igh

sa

lt bu

ffer

2.

WA

SH

: wa

sh

less h

yd

rop

ho

bic

pro

tein

s fro

m

co

lum

n in

med

ium

sa

lt bu

ffer

3.!

EL

UT

E: re

move (e

lute

) GF

P fro

m c

olu

mn

with

ve

ry lo

w s

alt b

uffe

r

99

Ste

p 1

: H

ydro

phobic

Inte

ractio

n C

hro

mato

gra

phy

!!A

dd b

acte

rial

lysate

to c

olu

mn

matrix

in h

igh

salt b

uffe

r "!H

ydro

ph

obic

pro

tein

s in

tera

ct

with

(bin

d to

) colu

mn

!!W

ash

less h

ydro

phobic

pro

tein

s fro

m c

olu

mn

with

med

ium

salt

bu

ffer

"!L

ess h

ydro

pho

bic

E

. coli p

rote

ins fa

ll fro

m c

olu

mn

"!G

FP

rem

ain

s b

oun

d

to th

e c

olu

mn

Ste

p 2

: H

ydro

phobic

Inte

ractio

n C

hro

mato

gra

phy

!!E

lute

GF

P fro

m

colu

mn b

y a

ddin

g

very

low

salt b

uffe

r

"!G

FP

ch

an

ges s

ha

pe

, an

d n

o lo

ng

er b

ind

s to

hyd

ropho

bic

colu

mn

m

atrix

.

"!G

FP

is e

lute

d fro

m th

e

colu

mn

and

flow

s in

to

the

co

llectio

n tu

be.

Ste

p 3

: H

ydro

phobic

Inte

ractio

n C

hro

mato

gra

phy

Help

ful H

ints

: H

ydro

phobic

Inte

ractio

n C

hro

mato

gra

phy

!!

Ad

d a

sm

all p

iece

of p

ap

er

to c

olle

ctio

n tu

be

wh

ere

co

lum

n s

its to

insu

re

co

lum

n flo

w

!!

Re

st p

ipe

tte tip

on

sid

e

of c

olu

mn

to a

vo

id

co

lum

n b

ed

dis

turb

an

ce

w

he

n a

dd

ing

so

lutio

ns

!!

Dra

in u

ntil th

e m

en

iscu

s

is ju

st a

bo

ve

the

ma

trix

for b

est s

ep

ara

tion

100

Procedure You will start with two microcentrifuge tubes containing ~2 mL each of bacterial cell culture. In

both cultures, 2 mL of LB nutrient growth media containing arabinose and ampicillin were inoculated with a bacterial colony. One of these cultures was inoculated with a colony from the LB/amp/ara plate, and the other was inoculated with a colony from the LB/amp plate.

Before examining the two cultures under UV light, predict with your lab group whether each culture will glow green under UV light. Record your predictions in your lab notebook. Then, observe both bacterial cultures under UV light. Record your observations in your notebook. Were your predictions correct? Why or why not? Consult with your instructor if you aren’t sure.

Now, you will begin to harvest and lyse the bacterial cells. Lysis (to lyse) means “to break open.” In order to begin to purify the GFP protein, we first need to lyse, or break open the bacterial cells to release all of the cellular contents (including proteins).

Throughout the GFP purification process, observe each step with the UV light to “follow” the GFP glowing protein product as it is purified—note your observations at each step in your notebook. 1. Harvest the cells from the LB/amp/ara liquid culture by centrifugation. Spin the

culture in a microcentrifuge for 1 minute at maximum speed. You should have a pellet of bacterial cells (if not, show your instructor before proceeding).

2. After centrifugation, open the tube and slowly pour off the liquid supernatant

into a waste beaker containing bleach. Observe the pellet under the UV light. Note your observations.

3. Add 250 µL of TE solution to the tube. Resuspend the bacterial pellet by pipetting up and down

with the P-1000 blue tip until there are no cell clumps. 4. Add 60 µL of lysozyme (obtain from instructor) to the resuspended bacterial cells. Cap and mix

the contents by flicking the tube with your index finger. Lysozyme is an enzyme that breaks down bacterial cell walls by digesting a polysaccharide in the cell wall. Lysozyme is naturally found in human tears, acting as a bactericidal agent to prevent eye infections. The enzyme gets its name from its ability to “lyse” bacteria.

5. Label your tube and place it in a rack in the freezer until next week. The freezing step is

important to complete lysis of the bacteria, because freezing will make the liquid inside bacterial cells expand, causing the cells to explode and break open completely.

101

Determining the Wavelength of Maximum Light Absorbance. The lambdamax for this sample is 440 nm. Once lambdamax is determined for a molecule, the spec is set to that wavelength, and all readings for the molecule are made at that wavelength.

Activity 11b Using the Spectrophotometer to Study Protein Concentration Purpose

In this activity, you will use a protein indicator called the Bradford reagent to visualize protein molecules in solution. You will determine the lambdamax (the wavelength of light with the maximum absorbance) for a protein solution containing the Bradford reagent using the spectrophotometer. This will then allow us to do Activity 11c.

Background

Proteins, like other molecules, interact with light waves and absorb or transmit light energy of various wavelengths. If set at an appropriate wavelength, a spectrophotometer can detect proteins in solution as the protein molecules absorb light energy. The more protein molecules in solution, the greater the absorbance should be.

Protein molecules are colorless. When dissolved in solution, they absorb light only in the UV range (specifically at ~200 and at 280 nm). Measuring protein concentration in the UV range, can however, be complicated by a number of factors that can make it difficult to consistently and accurately determine protein concentration. A more commonly used method that provides better accuracy and consistency is the Bradford protein assay. The Bradford assay uses a colored indicator, Bradford reagent. The Bradford reagent reacts proportionally with the protein to give a blue molecule.

To determine the lambdamax for our protein + Bradford reagent mixture, we must produce an absorption spectrum for the Bradford-protein pair and see where the peak of light absorption is. The lambdamax will then be used to detect protein molecules in solution in future experiments.

Procedure 1. Prepare 10 mL of a 20 mg/mL

BSA (bovine serum albumin) solution using your 50 mM Tris, 5 mM CaCl2, pH 7.2 solution from Activity 4b (week 4 of the semester). You will also use this buffer for all further dilutions and all “blanks.”

102

2. Using your Tris/CaCl2 buffer, make a 1:10 dilution of your BSA protein solution to obtain 5 mL of a 2 mg/mL BSA protein solution.

3. Preparation of sample tubes (your instructor will let you know which type of spectrophotometer is available)

If using VIS spectrophotometer with glass cuvettes: • Prepare a sample tube by placing 60 µL of a 2 mg/mL BSA solution into a glass cuvette. Add 3 mL of 1X Bradford reagent. Mix and let stand for ~ 3 minutes. • Prepare a blank by placing 60 µL of buffer into a glass cuvette. Add 3 mL of 1X Bradford reagent. Mix and let stand for ~ 3 minutes.

{Note: Please refer to spectrophotometer quick guide” on page 88 if using the VIS spectrophotometer to remind yourself how to use the spectrophotometer.}

If using UV/VIS spectrophotometer with glass cuvettes: • Prepare a sample tube by placing 20 µL of a 2 mg/mL BSA solution into a plastic cuvette. Add 1 mL of 1X Bradford reagent. Mix and let stand for ~ 3 minutes. • Prepare a blank by placing 20 µL of buffer into a glass cuvette. Add 1 mL of 1X Bradford reagent. Mix and let stand for ~ 3 minutes. 4. Measure the absorbance at 5 nm intervals from 560 nm to 620 nm, and record your data in a

table similar to Table 11.1 below. Table 11.1 Absorbance of Bradford-BSA mixture

Wavelength Absorbance 560 565 570 575 580 585 590 595 600 605 610 615 620

5. In your notebook, graph your absorbance data. Your graph will look similar to the one shown

on the previous page (p. 101). When you are finished with the graph, note the lambdamax in your notebook.

103

Activity 11c Determining the Concentration of BSA Protein in Solution Purpose

In this activity, you will use the Bradford assay method to determine the concentrations of two unknown BSA protein solutions.

Background

Often, the goal of a biotechnology company’s product pipeline is to manufacture a protein. The company must synthesize enough of the protein to market it at a profit. At every step in the manufacture of a protein, the concentration must be determined. Protein concentration is usually measured in milligrams per milliliter (mg/mL) or micrograms per milliliter (µg/mL). To determine the concentration of a solution, one produces a standard curve that plots the absorbance of solutions at known concentrations. First, the technician prepares solutions of known concentrations and reads their absorbance at a given wavelength. The technician then produces a “best-fit” straight line, representing how the concentration affects the absorbance by the molecule being tested. The absorbance of an unknown solution is then determined. From the “best-fit” straight-line standard curve, the concentration of the unknown solution is determined based on where the absorbance value intersects the standard curve (see figure).

Procedure 1. Using your 2 mg/mL BSA protein solution from Activity 11b and your Tris/CaCl2 buffer to dilute

the BSA protein solution, you will prepare 500 µL of each of the following solutions. Hint: you will need to use the dilution equation (C1V1 = C2V2) for these calculations.

• 1.6 mg/mL • 1.2 mg/mL

• 0.8 mg/mL

Standard Curve of Protein Concentration versus Absorbance. The absorbance of a sample is directly proportional to the number of molecules present (concentration). The “best-fit” line is used to determine the concentration of unknown protein samples.

104

• 0.4 mg/mL 2. Once you have made your known protein standards, obtain the two protein solutions of

unknown concentration from your instructor. 3. Label 8 glass or plastic cuvettes (depending on which type of spectrophotometer we will be

using—check with your instructor) as follows: (0 mg/mL (buffer), 0.4 mg/mL, 0.8 mg/mL, 1.2 mg/mL, 1.6 mg/ mL, 2 mg/mL, unknown A, and unknown B).

If using VIS spectrophotometer with glass cuvettes: • Prepare your samples by placing 30 µL of each solution into the appropriate labeled cuvette. Add 3 mL of 1X Bradford reagent. Mix and let stand for ~ 3 minutes.

If using UV/VIS spectrophotometer with glass cuvettes: • Prepare your samples by placing 10 µL of each solution into the appropriate labeled cuvette. Add 1 mL of 1X Bradford reagent. Mix and let stand for ~ 3 minutes. 4. Read each tube’s absorbance at the lambdamax that you determined in Activity 11b (check your

lambdamax with instructor). Record your data in a data table similar to Table 11.2 below: Table 11.2 Absorbance of protein standards and unknowns at lambdamax

Sample Absorbance at lambdamax 0 mg/ml (buffer)

0.4 mg/ml 0.8 mg/ml 1.2 mg/ml 1.6 mg/ml 2.0 mg/ml

Unknown A Unknown B

5. Using approximately a whole page of your notebook or graph paper, make a graph with sample

concentration (in mg/mL) on the X-axis and absorbance on the Y-axis. Make a “best fit” straight line of the known concentration samples versus their absorbance values.

6. Using this best-fit standard curve, estimate the concentration of Unknown A and Unknown B.

Show on your graph where the absorbance value for each unknown intersects the Y-axis, the “best-fit” straight line, and the X-axis (see the graph on p. 103 as an example).

7. Record the value for the concentration of amylase in Unknown A and in Unknown B in your

notebook. Check with your instructor to see how close you were to the actual concentrations of BSA in these protein solutions.

105

Lab 11 Homework Name: ________________________

1. Why was it necessary to add both arabinose and ampicillin to the LB nutrient growth medium in which the pGLO-transformed cells were inoculated? 2. When you examined the two bacterial cultures (one from the LB/amp/ara plate and one from the LB/amp plate) under the UV light, why were both cultures glowing green even though the bacterial colonies were not green on both plates? 3. Identify the function of these items used in today’s GFP purification procedure: a) Centrifuge b) Lysozyme c) Freezer 4. Why was it important to know the lambdamax for the Bradford reagent + protein solution? 5. Xanthophyll is the yellow pigment in lemons. What value would you expect the lambdamin to be for a xanthophylls solution. Why did you choose this value?