Westermeier R., Naven T., H?pker H.-R. Proteomics in Practice: A Guide to Successful Experimental Design

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Symptom Cause Remedy

DTT or other substances

such as SDS are present

in the labeling reaction

at too high a concentra-

tion.

Remove the substances

from the labeling reac-

tion if not essential. If

they are essential test if

the reduction in labeling

efficiency can be coun-

terbalanced by increas-

ing CyDye concentra-

tion.

The protein lysate con-

centration is too low, i.e.

less than 2 mg/mL.

Make a new batch of pro-

tein lysate reducing the

volume of lysis buffer to

increase the protein con-

centration. Or precipitate

the proteins and resus-

pend them in a smaller

volume of lysis buffer;

check the pH and con-

centration of the new

sample before labeling.

Vertical streaks and/or

vertical spot arrays

mainly in the basic area.

Multiple lysine labels

caused by overlabeling,

too high CyDye/protein

ratio.

Reduce amount of

CyDye, increase amount

of proteins, and improve

protein quantification

procedure.

Problems specifically with

saturation labeling

(cysteine labeling)

Spots shifting in hori-

zontal direction.

Overlabeling, too high

label/cysteine ratio in

the particular sample

type.

Decrease amount of

reductant and CyDye for

this particular sample

type. Optimize proce-

dure with same–same

experiment.

Spots elongated in verti-

cal direction and vertical

streaking.

Underlabeling, too low

label/cysteine ratio in

the particular sample

type.

Increase amount of

reductant and CyDye for

this particular sample

type. Optimize proce-

dure with same–same

experiment.

Trouble Shooting

420

Symptom Cause Remedy

Dark fluorescent back-

ground.

Too much Bind-Silane

applied.

Work strictly according

to the instruction and

remove excess of Bind-

Silane with lint-free tis-

sue.

Many dust particles. Powder from gloves. Use only powder-free

gloves.

Fluorescent streaks and/

or areas not correlated to

the front.

Contaminations with the

ink of a pen.

Do not write on cassettes

or casting box with a

pen, also not with a

waterproof pen.

The IPG strips appear

with high background.

The cassette has been

scanned with the film-

support of the IPG strip

down.

Apply the cassette with

the correct orientation,

in spite of the possibility

of correcting the image

later by flipping it with

the image analysis pro-

gram.

1.6

Results in 2-D Electrophoresis

Symptom Cause Remedy

No or very few spots. Low protein content. Check with reliable

quantification method;

check your entire proce-

dure with a standard,

like E. coli lyophylisate;

try alternative sample

extraction procedure.

Proteins have formed

complexes and did not

migrate into the gel.

Check sample prepara-

tion procedure, apply

cleanup based on precip-

itation.

1 Two-dimensional Electrophoresis

421

Symptom Cause Remedy

Problem(s) with silver

staining.

Check, whether all the

solutions have been

made correctly, the time

for each step has been

correct, and no step has

been forgotten. Do not

use plastic trays, but

glass or stainless steel.

Horizontal line across

the gel a few cm below

the upper edge with no

or blurred protein spots

between the upper edge

and the line.

Depletion of glycine. Use 2 SDS Tris-glycine

buffer in the upper buff-

er tank, fill enough buff-

er into the upper buffer

chamber

Missing protein spots in

the high molecular

weight area.

High molecular weight

proteins did not enter

the IPG strip.

Perform active rehydra-

tion with 50 V applied to

the IPG strip for 12

hours.

High molecular weight

proteins formed aggre-

gates in the first phase of

isoelectric focusing.

Apply “worst case condi-

tions” during isoelectric

focusing, which means

lower starting voltages

over longer time periods.

Equilibration of IPG

strip was too short.

Increase equilibration

steps to 2 20 minutes

Equilibration of IPG

strips was not efficient.

Increase SDS concentra-

tion in the equilibration

buffer to 6% (w/v).

Poor transfer of proteins

from the IPG strips to

the SDS gel.

Prolong the protein

transfer phase at low

power setting before you

switch to the separation

conditions.

Missing proteins. Sample application on

IPG strips was not opti-

mized.

Try all alternatives, also

cup loading on different

sides.

Equilibration was not

efficient enough.

Increase SDS content in

equilibration buffer from

2% to 6%.

422 Trouble Shooting

Symptom Cause Remedy

Vertical streaking. Dirty glass cassettes. Clean glass plates thor-

oughly, touch them only

with gloves.

Inefficient equilibration

of the IPG strip.

Equilibrate at least 2 15

minutes. Use iodoaceta-

mide in the second equi-

libration step.

Electroendosmotic

effects during first phase

of SDS electrophoresis.

Prolong the protein

transfer phase at low

power or current setting

before you switch to the

separation conditions.

Equilibration under oxi-

dative conditions.

Equilibrate first for 15

minutes in presence of

DTT and then 15 min-

utes in presence of

iodoacetamide.

Spots elongated in the

vertical direction.

Depletion of the upper

buffer, because of insuf-

ficient volume.

Fill enough buffer into

the upper buffer cham-

ber: 1.2 L into the Ettan

DALT 6 and 2.5 L into

the Ettan DALT 12.

Depletion of the upper

buffer

Use 2 SDS Tris-glycine

buffer in the upper buff-

er chamber.

Poor quality of chemi-

cals.

Use only high-quality

chemicals. In case of

doubt try reagents from

an alternative supplier.

High glycoprotein con-

tent.

Use gradient gels and

Tris-borate in the catho-

dal chamber instead of

Tris-glycine.

Vertical streak and

blurred pattern in the

acidic area, when

thiourea was used in the

first dimension.

Contaminated reagent. Try another batch of

thiourea.

4231 Two-dimensional Electrophoresis

Symptom Cause Remedy

Focusing of thiourea to-

gether with some other

reagents.

Reduce the volt-hours

applied to the IPG strip.

Follow the instructions

supplied with the IPG

strips, sometimes apply-

ing lower volt-hours.

Vertical perturbation in

the center of the gel, par-

ticularly in gels cast close

to the back of the six-gel

caster.

Heat development in the

solution feeding channel

during polymerization

causes local modifica-

tions in gel structure.

Pre-cool the monomer

solution in the refrigera-

tor.

Blurred or twinned

spots.

Gel strengthener was

used, gel composition is

modified.

Be careful when using

gel strengtheners.

Vertical gap(s). Air bubbles between first

and second dimension

gel.

Place the IPG strip care-

fully on the SDS gel and

seal it carefully with

agarose.

Amphoteric buffer in the

cell culture, for instance

HEPES, occupies a part

of the pH gradient.

Avoid amphoteric buf-

fers.

Double spots in vertical

direction.

IPG strip not applied in

the correct way.

Plastic film has to be in

contact with the glass

plate.

Multiple spots in vertical

direction in preparative

gels.

Insufficient DTT

amount.

Increase amount of DTT

during the first equili-

bration step.

Clouds of spots in the

low M

area.

Proteins partly digested

into peptides.

Add protease inhibitors

during protein extrac-

tion; use cup loading,

paper bridge loading, or

active rehydration load-

ing under voltage. Treat

the sample with a

cleanup procedure based

on precipitation.

Horizontal streaking. Particles in the sample

solution.

Always centrifuge the

sample before applica-

tion on the IPG strip. If

necessary, prolong cen-

trifugation.

424 Trouble Shooting

Symptom Cause Remedy

Underfocusing. Prolong the volt-hours in

the last IEF step.

Overfocusing: labile pro-

teins are degrading at

their pI. This happens

particularly in the basic

range.

Shorten the volt-hours in

the last IEF step.

Urea and/or detergent

concentration too low.

Increase concentrations

of urea to 9 mol/L and

CHAPS to 2% or 4%.

Wrong precipitation pro-

cedure was applied.

Do not apply ammo-

nium sulfate precipita-

tion. Either use cleanup

kit based on precipita-

tion or apply the proce-

dure according to Wes-

sels and Flgge (1984)

(methanol/chloroform).

Incomplete rehydration

of the IPG strip.

Check the rehydration

conditions.

TCA was not completely

removed from pellet

after precipitation

cleanup.

Add two or three wash-

ing steps with ice-cold

10% water/90% acetone.

Instability of some pro-

teins because of wrong

sample application.

Try alternatives, like cup

loading at the acidic or

basic end of the IPG

strip.

Highly abundant pro-

teins formed ridges and

were squeezed out, and

then became distributed

over the surface.

Run IPG strips contain-

ing highly abundant pro-

teins only with gel sur-

face up.

Horizontal streaking in

the acidic area.

Nucleic acids in the sam-

ple.

Apply a cleanup proce-

dure based on precipita-

tion. Alternatively add

benzonase or DNAse

and RNAse before add-

ing the urea (and

thiourea).

4251 Two-dimensional Electrophoresis

Symptom Cause Remedy

Horizontal streaking in

the basic area.

DTT depletion in the ba-

sic part of the gradient.

Pre-rehydrate the strips

with DeStreak solution

and apply reduced pro-

teins via cup-loading on

the anodal side.

Samples prepared with

DeStreak solution or

reagent.

Never treat the sample

with DeStreak. Treat

samples with a reduc-

tant. DeStreak is only

used in the IPG strip,

not in the sample.

DeStreak and reductant

has been mixed, result-

ing in the formation of

2-mercaptoethanol.

Never mix DeStreak and

a reductant, see above.

Horizontal streaking in

the acidic area and the

basic areas with good

separation in the center.

Salt content is too high. Treat the sample with

microdialysis or cleanup

with precipitation.

Electroendosmosis

effects at the ends of

IPG strips. IPG strips

have not been embedded

with agarose.

Always embed IPG

strips with agarose; this

equalizes the differences

of charged groups in the

IPG strip.

Electroendosmosis

effects at the ends of nar-

row interval IPG strips

because of accumulated

proteins of higher and

lower pIs.

Place filter paper pads

soaked in deionized

water between electrodes

and gel during the last

IEF step.

Horizontal streaking in

the center with good sep-

aration in the acidic area

and the basic areas.

Uneven rehydration of

the IPG strip, incom-

plete rehydration in the

middle.

During rehydration of

the strip, take care that

the liquid is distributed

evenly over the entire

strip length. Alterna-

tively, use a vertical rehy-

dration cassette for rehy-

dration of IPG strips.

426 Trouble Shooting

Symptom Cause Remedy

Protein spots arranged

like a string of beads in

horizontal direction.

Differential carbamyla-

tion of some proteins

because of presence of

isocyanate.

Do not heat sample. Do

not store urea and

thiourea solutions at

room temperature. Use

only high quality urea

and thiourea. Remove

ionic compounds with

mixed bed ion exchan-

ger.

Storage proteins in plant

seed extracts, differen-

tially glycosylated.

No artifact. Nothing to

worry about.

Cloudy background and/

or front.

Micelles between SDS

and the nonionic or zwit-

terionic detergent have

formed.

Reduce content of

CHAPS or alternative

detergent in the rehydra-

tion solution for the IPG

strip (e.g. maximum 2%

CHAPS).

Blurred and streaky spot

pattern in preparative

gels.

Insufficient IEF condi-

tions.

Prolong the volthours in

the last IEF step by 15%.

DTT depletion in prepar-

ative basic gels.

Add 2.5% DTT, 20% iso-

propanol, 5% glycerol to

the rehydration solution,

apply paper wick soaked

in rehydration solution

plus 3.5% DTT at the

cathode. Or apply

DeStreak concept (see

above).

Blurred spot pattern in

ready-made gels.

Liquid and/or bubbles

between surface of

ready-made gel and glass

plate.

Use only low amount of

gel buffer and roll out

excess buffer and air

bubbles completely.

Very basic proteins are

lost.

Too many volt-hours

applied on basic gradi-

ents, autohydrolysis of

basic buffering groups

in the gel.

Shorten the volt-hours in

the last IEF step. Apply

pH 7–11 NL strip, this

one is stabilized against

hydrolysis.

4271 Two-dimensional Electrophoresis

Symptom Cause Remedy

,,Wrong” proteins com-

ing from other organ-

isms, which should not

be present, have been

identified in the gel.

Contamination with pre-

vious sample or proteins

from the laboratory envi-

ronment.

Use only highly pure

reagents; clean equip-

ment thoroughly, partic-

ularly reswelling tray

and strip holders for IPG

strips; filter solutions

through a membrane fil-

ter.

428 Trouble Shooting

Symptom Cause Remedy

No signal in reflectron

mode MALDI MS.

No accelerating voltage. Check read out from

electronics gate.

Laser not firing. Contact supplier.

Laser not firing in cor-

rect place.

Re-alignment of laser re-

quired.

Laser power far too low. Increase laser power.

Potentially a detector or

amplifier problem.

Check signal in linear

mode.

Poor analyte signal. Insufficient protein pres-

ent in gel plug for diges-

tion.

Run a new gel with a

higher protein load.

Trypsin inactivity. Prepare trypsin fresh on

ice. Perform rehydration

step on ice. Ensure cor-

rect buffer for trypsin

activity.

Sample concentration

too low.

Use 0.1–10 pmol/mL

final concentration.

Sample concentration

too high.

Sample signal may be

suppressed. Dilute sam-

ple to 0.1–10 pmol/mL

final concentration.

Poor crystallization,

presence of salt/buffer.

Use a higher concentra-

tion on TFA (up to 1%)

to improve ionization.

Avoid phosphorylated/

sulfated buffers.

4292 Mass Spectrometry

Mass Spectrometry