Williams 11 - Varia e Biblio.pdf - Książki - neo666x

69, 372-384

(1975)

ANALYTICAL

BIOCHEMISTRY

Electrophoresis

with

Continuous

Scanning

Densitometry:

Separation

of Cells

in a Density

Gradient

N. CATSIMPOOLAS, A. L. GRIFFITH,

AND J. M. WILLIAMS

Biophysics Laboratory,

Department

of Nutrition

and Food Science,

Massachusetts

Institute

of Technology, Cambridge, Massachusetts

02139

A. CHRAMBACH AND D. RODBARD

Reproduction

Research Branch, National

Institute

of Child Health and Human

Development, National

Institutes of Health, Bethesda, Maryland

20014

Received March

21, 1975; accepted May 22, 1975

An instrument and procedure for electrophoresis with continuous optical scan-

ning densitometry, automated data processing, and related methodology are de-

scribed for the continuous analysis of electrophoresis and unity gravity sedi-

mentation of macromolecules or cells in a static density gradient system. The

instrument consists of a dual-beam spectrophotometer, a scanning stage and

scanner control unit, an electrophoresis cell cassette, a filling/purging/cooling

module, an analog-to-digital converter, and a digital data-logger. The distribution

of cells is monitored repetitively during migration by absorbance measurements at

any wavelength in the 200-800-nm range. A computer program provides the

statistical analysis of each peak (baseline correction, smoothing, area, mean, stan-

dard deviation, skewness, and kurtosis) which can be further utilized for com-

puting additional parameters, such as resolution and heterogeneity. A mixture of

human and rabbit erythrocytes were used as a model system to evaluate the per-

formance of the instrument and demonstrate some of its capabilities.

A considerable

body of knowledge

about the electrokinetic

behavior

of cells has been acquired

by the microscope

electrophoresis

technique

(1,2X

However,

bulk

separation

and analysis

of cell populations

electrophoretic

methods

has been limited

primarily

to flow methods,

including

free-flow electrophoresis

(3,4), steady-flow

(STAFLO)

elec-

trophoresis

(5), and endless

belt electrophoresis

(6). The

use of sta-

tionary

vertical

density

gradients

for the stabilization

and preparative

electrophoresis

of viable

mammalian

cells was introduced

only recently

(733).

In the present

study, fractionation

of cells was carried

out in an in-

strument

capable

of repetitive

optical

scanning

during

electrophoresis.

The instrument

is a modification,

largely

based on commercially

avail-

able components,

of previous

designs (9) which are similar

to devices

372

1975

by Academic

Press, Inc.

All

rights of reproduction

in any form reserved.

373

SCANNING

ELECTROPHORESIS

OF CELLS

used in gel filtration

(10) and “free

zone electrophoresis”

(11). These

devices, in turn, are descendants

of the original

Tiselius

apparatus.

The application

of the continuous

scanning instrument

to cell separa-

tion provides

analysis of physical-chemical

properties,

such as average

velocity,

mobility,

charge heterogeneity,

and resolution

in addition

cell separation.

Human

and rabbit

erythrocytes

were selected for frac-

tionation

because they exhibit

a rather large electrophoretic

mobility

dif-

ference of at least 0.6 X 1O-5 cm2/sec/V

(in 0.15 M saline,

7.2, at

25°C) by the microscope

method

(2). At its present

state of develop-

ment, the instrument

described here must be considered

analytical

rather

than preparative

since it is capable of handling

a maximum

of 106-lo7

cells per experiment.

However,

collection

of fractions

for further analy-

sis is possible.

APPARATUS

The principles

of electrophoretic

analysis

with continuous

scanning

during electrophoresis

have been reviewed

previously

(9). A schematic

diagram

of an improved

apparatus

and accessories is provided

in Fig. 1.

The dual-beam

spectrophotometer

is similar

to that described

previously

(12) except for the following:

A 200-W xenon-mercury

arc lamp and as-

sociated power supply (Schoeffel) are used in conjunction

with a tandem

grating

monochromator

(Schoeffel

GMIOOD)

to produce

monochro-

matic light in the 200-700-nm

wavelength

range of very low stray light

characteristics

(specified

by the manufacturers,

1: IO4 at 220 nm) (Fig.

2). Slit widths available

for each monochromator

are 0.5, 1.0, 1.4, 2.0,

PHOTO-

SPECTRCOENSlTO-

LAMP

MULTI

METER

PLIERS

FILLING/PURGING

RECORDER

MODULE

;------

--------__

8 REMOTE

COMPUTER

t _____-_-

- --_--_-

1. Block diagram of the apparatus.

FIG.

374

CATSIMPOOLAS

AL.

2. Schematic diagram of the spectrophotometer

scanning stage.

FIG.

and 2.5 mm. The monochromatic light beam is subsequently collimated

by a quartz lens, shaped by a variable horizontal slit (25-100 pm), and

divided to illuminate the sample and reference electrophoresis cells

simultaneously. Each beam is detected separately by its own photomul-

tiplier tube, and the log of the ratio of the two signals, i.e., the linear op-

tical density, is obtained

electronically

(Schoeffel

model

SD 3000 Spec-

trodensitometer).

The use of a split-beam

instrument

eliminates

errors

due to fluctuations

of the power supply,

light

intensity,

and photomul-

tiplier

high voltage.

A balance control

and zero meter permit

matching

sensitivity

levels of the photomultipliers

at any desired wavelength.

This

adjustment

equilizes

the electrical

outputs of the two channels, providing

a reading of zero optical

density (OD) at the beginning

of the scan, with

the sample beam in a “neutral”

area of the media

to be measured.

The

photometer

supplies an analog output of 1 V per 1 OD unit and 100 mV

scale (corresponding

to 0.1, 0.2, 0.4, 1, 2, 4, or 10 OD units)

for

operating

a recorder.

The output

is processed

by an analog-to-digital

converter

and recorded

on perforated

paper tape. An optional

single

channel

mode of operation

is also available

in the instrument.

SCANNING STAGE

Linear trunsport:1 This part of the instrument

provides

vertical

linear

transport

of a stage for the removable

electrophoresis

cassette by means

of a reversible

stepping motor attached to a precision

leadscrew (Fig. 3).

The

stepping

motor

and adjustable

frequency

generator

provide

six

1 Blueprints,

engineering

drawings, or sample output provided upon request.

375

SCANNING

ELECTROPHORESIS

CELLS

Rubber

Washer

Cooler

Block

Lower

Elect

3. Schematic diagram of the electrophoresis

cassette.

FIG.

reversible up or down scanning speeds of 5, 10, 20, 40, 80, and

160 se&m. This unit also provides dark housing.

Control unit? The control unit regulates the movement of the scan-

ning stage, the X-Y recorder, and the tape puncher. A 25kohm

“scanner pickoff” linear displacement potentiometer is mechanically

fixed to the moving stage. The potentiometer is driven from an appropri-

ately scaled dc voltage source such that the output voltage is equal to

the position of the scanner (in centimeters) with 0.05% accuracy. This

potential is displayed on a digital panel meter (Newport 2000) which

provides a direct readout of the scanner position in centimeters. The

signal is also supplied to two amplifiers which are in a double limit com-

parator configuration. Two 10-kohm ten-turn potentiometers permit

setting up “high’ and “low” scanner travel limit positions and are

calibrated directly in centimeters with a 1% accuracy. The comparator

continuously compares the limit potentiometer settings with the pickoff

signal and issues motor-reversing commands to the memory/switching

element which functions as a set-reset flip-flop, with versatile switching

capability and high noise immunity. One set of relay contacts functions

as a latch and provides the “memory” function. A second set of contacts

transmits the reversing information to the motor. A third set of contacts

automatically operates the “sweep” and “reset” functions of an X-Y

recorder and signals the paper tape punch to issue a leader (blank

space

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