2D电泳(常见问题与原因)

283

1

Two-dimensional Electrophoresis

Additional trouble shooting guides for 2-D electrophoresis are found in:

.

Berkelman T,Stenstedt T.Handbook:2-D electrophoresis using immobilized pH gradients.Principles &methods.Ame-rsham Biosciences 80-6429-60(1998).

.

Görg A,Weiss W.Two-dimensional electrophoresis with immobilized pH gradients.In Rabilloud T,Ed.Proteome research:Two-dimensional gel electrophoresis and identifica-tion methods.Springer Berlin Heidelberg New York (2000)107±126.

In the following part only a selection of cases are described,which typically occur now and then.

1.1

Isoelectric focusing in IPG strips

Rehydration solution is distributed unevenly within the gel strip.

Some coating in the strip holder or reswelling tray.Wash strip holder and res-welling tray with

detergent,rinse with deionised water.

Uneven pipetting of the rehydration solution.Pipette the solution as a streak.Reswelling tray or IPGphor not levelled.Adjust the level of the res-welling tray or the

IPGphor on the bench.Rehydration liquid is left in the reswelling tray or strip holder.

Rehydration time too

short.

Rehydrate at least for 6hours without and 12hours with sample.A Trouble shooting

Proteomics in Practice:A Laboratory Manual of Proteome Analysis

Reiner Westermeier,Tom Naven

Copyright 2002Wiley-VCH Verlag GmbH

ISBNs:3-527-30354-5(Hardcover);3-527-60017-5(Electronic)

Including images of bad gels.

Liquid volume too high.Follow the recommendati-

ons on the package.

IPG strips improperly sto-red.Always store IPG strips in the freezer,do not leave them on the bench at room temperature for too long time.

Basic part of the gel comes off during rehydration.Surface has been

damaged during removal

of cover film

Always start at the acidic,

pointed side to remove the

cover film.

Voltage too low(8kV not reached)Short strips(7cm and

11cm)are used,8kV is

reached in those strips

only with some samples

under exceptional

conditions.

Nothing to worry about.

Poor quality of urea and/

or thiourea.

Use high quality urea and

thiourea,remove ions with

a mixed bed ion exchan-

ger.

Too much salt in the

sample.

Remove salts by micro-

dialysis or precipitation,

replace PBS for cell wash-

ing with something non-

ionic.

TCA left in the sample

from precipitation.

Add more washing steps.

Bromophenol blue band stops and does not migrate completely into the anode.Too much salt in the

sample.

See above.

Strip starts to burn at a certain position.Too much salt in the

sample.

See above.

Visible brown band develops.Tris-chloride in the

sample and rehydration

solution.

Run the sample in a cup-

loading strip holder.

Cover fluid(paraffin oil) leaks out of the cup loa-ding strip holder during IEF.High protein and salt

load cause water

transport that carries the

oil with it.

Reduce the initial voltage

and prolong the first low-

voltage steps.

284A Trouble shooting