133

Part II:

Equipment and methods

Electrophoresis in Practice, Fourth Edition. Reiner WestermeierCopyright � 2005 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 3-527-31181-5

135

Almost all methods described here are performed in a horizontal sys-tem with the same equipment. In methods10 and 11also proceduresfor vertical equipment is explained.

In principle the sequence of the first nine methods does not corre-spond to their importance or frequency of use but rather their sim-plicity and cost. The DNA methods are placed to the end, because allof them are new developments.

Methods:

Small molecules:

1. PAGE of dyes4. Native PAGE in amphoteric buffers

Proteins:

2. Agarose and immunoelectrophoresis3. Titration curve analysis4. Native PAGE in amphoteric buffers5. Agarose IEF6. PAGIEF in rehydrated gels7. Horizontal SDS PAGE8. Vertical PAGE9. Semi-dry blotting of proteins

10. IEF in immobilized pH gradients11. High resolution 2-D electrophoresis

Equipment for part II

Electrophoresis in Practice, Fourth Edition. Reiner WestermeierCopyright � 2005 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 3-527-31181-5

The small items and piecesof equipment as well as theprincipal stock solutionscan be used for almostall the methods.

This should also help for theplanning of an electrophoresiscourse.

Equipment for part II

DNA:

8. Vertical PAGE12. PAGE of double stranded DNA13. Native PAGE of single stranded DNA14. Denaturing gradient gel electrophoresis15. Denaturing PAGE of DNA

InstrumentationMultiphor II horizontal electrophoresis unitMighty Small SE 250 vertical electrophoresis unitEPS 3501 XL programmable power supply 3500 VMultitemp III thermostatic circulatorProcessor Plus silver staining and blot developing

apparatusNovablot graphite plate electrodes for blottingFilm Remover apparatus for removing support filmsIPG strip kit for IPG-IEF of 2D electrophoresis

on MultiphorStrip positioner for SDS PAGE on MultiphorIPGphor, strip trays IEF system for 2-D electrophoresisEttan DALT II SDS PAGE system for 2-D electrophoresisGel dryer gel drying frames and loading platformUniversal gel kit: contains glass plates, glass plates with

gaskets, clamps, gradient mixer, tubing,tubing clamps, scalpel, tape;

GelPool Tray for rehydration of dry gelsPaperPool Double tray for soaking of electrode wicksImmmuno-electrophoresis kit: contains levelling table, spirit level, glass

plates, electrode wicks, holder, specialscalpell, punching template, gel punchwith vacuum tubing, sampleapplication film;

field strength probe and voltmeterhand roller,water gaugesilicone rubber sample applicator maskhumidity chamber for the agarose techniquesstainless steel staining traysdestaining tanksglass tray for silver staining

136

for methods 1 to 9, 11

only for method 9!

Equipment for part II

scissors, spatulasassorted glass ware: beakers, measuring cylinders, erlenmeyers,test tubes etc.magnetic stirrer bars in different sizesgraduated pipettes of 5 and 10 mL + pipetting device(e.g. Peleus ball)micropipettes adjustable from 2 to 1,000 lL

Consumables:

Disposible gloves, tissue paper, filter paper, Scotch and Dymo tapepipette tips, Eppendorf cups, test tubes with screw caps 15 and

50 mL

Evaluation:

ImageScanner desk top scanner modified for electrophoresisgels

Typhoon multifluorescence scannerComputer (Windows)ImageMaster 1 D and 2D evaluation software

137

138 Equipment for part II

SPE

CIA

LL

AB

OR

AT

OR

YE

QU

IPM

EN

TM

etho

d1

23

45

67

89

1011

1213

1415

Gla

ssro

dm

m

Hea

ting

bloc

kp

pp

p

Hea

ting

cabi

neto

rin

cuba

tor

mm

mn

mm

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ting

stir

rer

nn

nn

n

Lab

orat

ory

elev

ator

(“L

abor

boy”

)m

mm

mm

Mic

row

ave

oven

mm

m

Pap

ercu

tter

“Rol

land

Cut

”.m

Roc

king

plat

form

mn

mn

nmn

mn

mn

mn

n

Smal

lmag

neti

cst

irre

rm

mm

mm

Spat

ula

indi

ffer

ents

izes

mm

mm

mm

mm

mm

mm

mm

m

Tabl

ece

ntri

fuge

pp

pp

pp

pp

pp

pp

p

Forc

eps

stra

ight

and

curv

edm

mm

mm

mm

m

UV

lam

pn

Ven

tila

tor

mm

mm

mm

mm

m

Wat

chgl

asse

sm

m

Wat

erje

tvac

uum

pum

pm

mm

mm

m

pFo

rsa

mpl

epr

epar

atio

n,m

for

the

met

hod,

nfo

rde

tect

ion

139Equipment for part II

CO

NSU

MA

BL

ES

Met

hod

12

34

56

78

910

1112

1314

15

Blo

ttin

gm

embr

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nitr

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lulo

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loph

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nn

Dym

ota

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mm

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mm

m

Ele

ctro

dew

icks

mm

mm

mm

mm

m

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per

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mm

mm

mm

mm

Focu

sing

stri

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mm

m

Gel

Bon

d�Fi

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rag

aros

e(1

2.5

�26

cm)

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lm(1

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mm

mm

mm

mm

mm

m

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Bon

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cm)

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mm

m

Pip

ette

tips

pm

pm

pm

pm

pm

pm

pm

pm

pm

pm

pm

pm

pm

pm

Pla

stic

bags

mm

mm

mm

mm

mm

m

Poly

este

rfi

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trea

ted

mm

mm

mm

mm

mm

PV

Cfi

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verh

ead)

n

Sam

ple

appl

icat

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mm

pFo

rsa

mpl

eap

plic

atio

n,m

for

the

met

hod,

nfo

rde

tect

ion

140 Equipment for part IIC

HE

MIC

AL

SC

hem

ical

sM

etho

d1

23

45

67

89

1011

1213

1415

Ace

tic

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(96

%)

nn

mn

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mm

mm

mm

mm

mm

mm

Aga

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Lm

m

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m

Mix

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mm

mm

mm

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mm

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),N

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ate

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nn

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mm

mm

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mm

mm

m

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pend

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Bor

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cium

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ate

m

pFo

rsa

mpl

epr

epar

atio

n,m

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the

met

hod,

nfo

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tect

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141Equipment for part IIC

HE

MIC

AL

S,C

ON

TIN

UE

DM

etho

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mp

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anol

nn

nn

nn

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Eth

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glyc

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mm

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amid

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nn

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Gly

cero

lm

mm

mm

mm

mm

mm

m

Gly

cine

nn

nn

nm

mm

mm

nn

nn

142 Equipment for part II

CH

EM

ICA

LS,

CO

NT

INU

ED

Met

hod

12

34

56

78

910

1112

1314

15

HE

PE

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umin

gm

mm

m

Imm

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�10

mL

)m

m

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Ker

osen

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gm

mm

mm

mm

mm

mm

mm

m

Mar

ker

prot

eins

for

MW

Cal

ibra

tion

kit(

1440

0to

9400

0D

a)m

mm

Col

lage

nso

lutio

nup

to30

0kD

am

m

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tide

mar

ker

mm

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for

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ln

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nn

n

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143Equipment for part IIC

HE

MIC

AL

S,C

ON

TIN

UE

DM

etho

d1

23

45

67

89

1011

1213

1415

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tor

pp

pp

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rmal

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e,de

pend

ing

onth

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mm

pp

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stG

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Blu

eR

,st

aini

ngta

blet

sC

oom

assi

eR

-350

nn

nn

nn

nn

Pho

spho

ric

acid

nn

nm

mm

m

PM

SF,p

rote

ase

inhi

bito

rp

pp

pp

Rep

elSi

lane

mm

mm

mm

mm

mm

mm

m

SDS

pm

pm

mpm

m

Silv

erni

trat

en

nn

nn

nn

nn

nn

Soda

lime

pelle

ts(C

O2

trap

)m

mm

m

Sodi

umac

etat

en

nn

n

Sodi

umca

rbon

ate

nn

nn

nn

nn

nn

n

Sodi

umch

lori

den

n

Sodi

umdi

hydr

ogen

phos

phat

em

n

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drog

enph

osph

ate

mn

Sodi

umhy

drox

idm

mn

mm

p

Sodi

umth

iosu

lpha

ten

nn

nn

nn

n

Sorb

itol

mm

m

144 Equipment for part II

CH

EM

ICA

LS,

CO

NT

INU

ED

Met

hod

12

34

56

78

910

1112

1314

15

Sulf

uric

acid

conc

.n

nn

Sulp

hosa

licyl

icac

idn

TE

ME

Dm

mm

mm

mm

mm

mm

m

Thi

oure

apm

TM

PT

MA

n

Tric

hlor

acet

icac

idn

nn

nn

nn

Tric

ine

mm

mm

mm

mm

Tris

mm

mm

mm

mm

mm

m

Tung

stos

ilici

cac

idn

nn

nn

Twee

n20

n

Ure

amp

mp

mp

pm

pm

Zin

csu

lpha

ten

nn

All

chem

ical

sm

ustb

eof

anal

ytic

al(p

.A.)

qual

ity.

Dou

ble-

dist

illed

wat

ersh

ould

beus

edfo

ral

lsol

utio

ns.

pFo

rsa

mpl

epr

epar

atio

n,m

for

the

met

hod,

nfo

rde

tect

ion