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

Подождите немного. Документ загружается.

281

Equipment, Consumables, Reagents

Sample Preparation

Instrumentation

Freezer –20 C, –80 C

Laboratory centrifuge for 1.5 mL tubes, at least 13,000 rpm

Heating block for 1.5 mL tubes, at least 95 C

Micropipettes adjustable from 2 mL to 1,000 mL

Optional (not always needed):

Sonicator probe

Consumables

Disposable powder free gloves

Reagents

Urea, thiourea, CHAPS, Dithiothreitol (DTT), Bromophenol blue,

SDS, Tris-base, Glycerol (85%), Pharmalytes pH 3–10 (~40% w/v),

IPG buffer,

Ettan 2D cleanup kit

Iodoacetamide

Optional (not always needed):

Molecular grinding kit,

PlusOne microdialysis kit

Ettan 2D quantification kit

Desoxyribonuclease I (DNase I)

Ribonuclease I (RNase A and RNase B)

Ribonuclease I “A” (RNase A)

Protease inhibitor mix

Phosphatase inhibitors: Na-vanadate, NaF

Sulfobetains

Proteomics in Practice. A Guide to Successful Experimental Design 2

Ed.

Reiner Westermeier, Tom Naven, and Hans-Rudolf Hçpker

ISBN: 978-3-527-31941-1

Equipment, Consumables, Reagents282

2-D DIGE

Instrumentation

Typhoon 9400, Trio multifluorescence and phosphoimaging

scanner

or:

Ettan DIGE Imager (EDI)

Reagents

Dimethylformamide

CyDyes for minimal labeling, lysine

CyDyes for saturation labeling, HCEP

2-D Electrophoresis

Instrumentation

IPGphor IEF system for 2-D electrophoresis

Regular strip holders

18 cm, 24 cm

for standard IEF in Immobiline DryStrips

Ceramic Manifold

24 cm

for basic gradients and very high sample

loads

Reswelling tray for rehydrating 12 IPG strips 7–24 cm

Ettan DALT twelve SDS PAGE system for high-throughput 2-D

electrophoresis

Ettan DALT six SDS PAGE system for medium-throughput

2-D electrophoresis

with:

Multitemp III thermostatic circulator

EPS 3501 XL programmable power supply 3,500 V

or:

EPS 600 power supply 600 V

Gel caster for 14 gels

Gel caster for 6 gels

Precast gel cassette 1.0 mm

Gel casting cassette 1.0 mm

Gel casting cassettes

low-fluorescence

(for DIGE gels)

1.0 mm

Gel casting cassette 1.5 mm

Blank cassette insert

Separator sheets 0.5 mm

Equipment, Consumables, Reagents 283

Filler sheets 1.0 mm

Cassette rack

Equilibration tube set 24 cm

Thin plastic ruler (300–500 mm)

Heating block for 1.5 mL tubes, at least 95 C

Magnetic stirrer

Laboratory shaker

Ring stand

Hand roller, scissors, spatulas, forceps with bent pointed tips

Assorted glassware: beakers, measuring cylinders, erlenmeyers, test

tubes etc.

Magnetic stirrer bars in different sizes

Squeeze bottle, finger-pumped plant sprayer (ca. 500 mL)

Graduated pipettes of 5 mL and 10 mL + pipetting device

(e.g. Peleus ball)

Micropipettes adjustable from 2 mL to 1,000 mL

Waterproof pen, black, like Edding 3000

Optional:

FilmRemover apparatus for removing gels from support

films

Gradient maker for multiple gel caster

Laboratory platform, adjustable, Pinchcock clamp

Consumables

Paper electrode strips, paper bridge pads, loading cups,

Disposable gloves, tissue paper, filter paper, pipette tips, microcentri-

fuge tubes, test tubes with screw caps 15 mL and 50 mL,

Reagents

IPG DryStrips, 18 cm or 24 cm, different pH gradients

DeStreak (HED)

IPG cover fluid

IPGphor strip holder cleaning solution (neutral detergent)

Ettan DALT II gels and buffer kit:

DALT II Gel 12.5% homogeneous 6/pk

DALT II buffer kit

Urea, thiourea, CHAPS, Dithiothreitol (DTT), Bromophenol blue,

SDS, Tris, Pharmalytes pH 3–10 (~40% w/v), IPG buffers

Equipment, Consumables, Reagents284

Acrylamide IEF solution (40%T,3%C), or Acrylamide PAGE 40%

solution, N,N¢-methylenebisacrylamide (Bis), Glycerol (85%),

Ammonium persulfate, TEMED, Glycine, GelSeal silicone grease,

Agarose NA

Molecular weight markers range 14,400–94,000

IEF sample application pieces

Gel labels cut from a printed paper or overhead film

Bind-Silane, Decon 90

Thiourea, alternative detergents, sulfobetains, isopropanol

Acid violet 17, phosphoric acid, trichloroacetic acid, imidazole, zinc

sulfate, EDTA-Na

Staining

Instrumentation

Processor Plus with

large tray

apparatus for automated silver staining

Staining kit with set

of trays

for multiple gel staining

Orbital shaker

Stainless steel staining trays (for Coomassie and silver staining)

Heating stirrer

Kitchen foil welding apparatus

Dark plastic tray (for Deep Purple)

Gel dryer gel drying frames and loading platform

Consumables

Disposable gloves

Transparent smooth sheet

protectors

A4 or letter format

Reagents

PhastGel blue tablets Coomassie brilliant blue R-350

Acetic acid

PlusOne silver staining kit

Coomassie brilliant blue G-250, Tris, o-phosphoric acid, ammonium

sulfate, methanol

Deep Purple

Equipment, Consumables, Reagents 285

Evaluation

Instrumentation

ImageScanner desk top scanner modified for electro-

phoresis gels

Typhoon 9000 multifluorescence and phosphoimaging

scanner

Ettan DIGE Imager (EDI)

Computer (Windows NT , 2000 or XP)

Printer

Software

DeCyder 2D evaluation software for DIGE gels

DeCyder EDA Extended data analysis with multivariate

statistical tools

ImageMaster

Platinum

2D evaluation software

Gray step tablet

Spot Picking

Instrumentation

Ettan spot picker

Consumables

Powder-free gloves

500 mL Eppendorff tubes

Scalpel

Surgical needles

Reagents

Ammonium bicarbonate

Acetonitrile

MilliQ water

Digestion

Instrumentation

Ettan digester

Equipment, Consumables, Reagents286

Consumables

Reagents

DDT

Iodoacetamide

Sequence grade trypsin

Ammonium bicarbonate

Trifluoro-acetic acid

Acetonitrile

MilliQ water

Mass Spectrometry

Instrumentation

Ettan MALDI-ToF Pro

Consumables

Ettan MALDI-ToF Pro target slides

Gel loader pipette tips

ZipTips

Reagents

a-Cyano-4-hydroxycinnamic acid

2,5 Dihydroxybenzoic acid

Heptafluorobutryic acid

RP resin

Trifluoroacetic acid

Acetonitrile

Methanol

Formic acid

MilliQ water

287

Step 1: Sample Preparation

To explain here all ways of sample preparation in detail would be too

much for this book. Therefore the following instructions should be

seen as a starting point. For instance very hydrophobic proteins or

plant proteins require more sophisticated extraction procedures.

Sample preparation is the most sensitive step in the entire proce-

dure. Proteins, which got lost here, cannot be identified. Modifica-

tions of proteins lead to wrong conclusions in proteome analysis.

To avoid protein losses, the treatment of the sam-

ple must be kept to a minimum.

To avoid protein modifications, the sample

should be kept as cold as possible.

To avoid losses and modifications, the prepara-

tion time should be kept as short as possible.

It should also be noted that there are more than one possible proce-

dure to treat a sample. A 100% representation of the proteins con-

tained in a cell will never be obtained in practice. Usually the method,

which will display the highest number of different proteins, is cho-

sen. When there is special interest in a certain group of proteins,

which are under-represented with the default method, an alternative

procedure has to be applied.

Example: When – for a given sample – the highest number of pro-

teins gets extracted under alkaline conditions, a number of basic pro-

teins will not be included, because their solubility close to their iso-

electric points is low. This means: For the analysis of the basic pro-

teins acidic extraction conditions have to be employed.

1.1

Washing of Cells

Washing of cells is a critical step, where salt ions can be introduced

into the sample. PBS is the standard washing solution. If it is not pos-

Proteomics in Practice. A Guide to Successful Experimental Design 2

Ed.

Reiner Westermeier, Tom Naven, and Hans-Rudolf Hçpker

ISBN: 978-3-527-31941-1

The procedure described for

basic proteins in this chapter is

based on a procedure devel-

oped for optimal extraction and

concentration of plant proteins.

Step 1: Sample Preparation288

sible to remove the PBS solution completely, it is better to wash the

cells with Tris-buffered sucrose (10 mmol/L Tris, 250 mmol/L

sucrose, pH 7), or to use this alternative washing solution as a last

washing step.

Tris-buffered sucrose washing solution: 1.21 g Tris, 85.6 g sucrose, fill

up to 1 L with H

dist

, titrate to pH 7 with 4 mol/L HCl.

First the default procedure and lysis solution is described (see

Tables 1.2, 1.3, 1.4). Alternative or additional reagents should only be

used, when the results are not sufficiently good.

The first dimension, isoelectric focusing, is relatively sensitive to

salts and other ionic contaminations. Thus the use of ionic buffers

must be avoided as much as possible.

The detection methods listed in Table 1.1 describe categories of

sample loads. For facts and comments on detection methods see page

50 and Step 4: Staining of gels.

Tab. 1.1: Protein loads (on large gels, using 18 cm and 24 cm strips).

Radiolabeled 1 mg–50mg “Analytical”

Silver stain 20 mg – 200 mg “Analytical”

CyDye labeled (lysine) 50 mg to 450 mg “Analytical to preparative”

CyDye labeled (cysteine) 500 ng – 300mg “Analytical to preparative”

Deep Purple stain 100 mg – 700 mg “Analytical”

Zinc imidazol negative stain 200 mg – 500 mg “Analytical”

Coomassie blue stain 500 mg – 2 mg “Preparative”

Coomassie blue stain 1 mg – 10 mg “Preparative” on narrow

pH intervals

The maximum applicable sample loads depend on the length of the

IPG DryStrip and the kind of pH gradient.

& Note: A sample for 2-D electrophoresis is very

precious, only high purity reagents should be

used.

1.2

Stock Solutions

Disposable gloves must be worn during each step to avoid contamina-

tion of the sample.

For the sake of reproducibility it is proposed to use exact concentra-

tions of Bromophenol blue.

Pharmalytes and IPG buffers

are amphoteric and acquire a

net charge of zero during IEF.

1.2 Stock Solutions 289

1% (w/v) Bromophenol blue solution:

Bromophenol blue 1% (w/v) 100 mg

Tris-base 60 mg

Water, deionized Dissolve 10 mL

Tab. 1.2: Standard solubilization cocktail “lysis solution, lysis buffer”

(10 mL).

9 mol/L urea 5.4 g

4% (w/v) CHAPS 400 mg

1% (w/v) DTT (= 65 mmol/L) 100 mg

2% (v/v) Pharmalytes pH 3 to 10 *) 200 mL

0.002% Bromophenol blue **) 10 mL

Water, deionized, make up to 10 mL

*) or IPG buffer of respective pH interval

**) from 1% Bromophenol blue solution

& Important: Prepare the solution freshly, shake to

dissolve the urea, do not warm it higher than

30 C to avoid carbamylation.

Removal of isocyanate

If the purity of the urea is in doubt:

Dissolve 5.7 g urea in deionized water, fill up to

10 mL.

Warm the tube to get the urea completely in

solution: Do not exceed 37 C.

Add 100 mg mixed bed ion exchanger Amberlite

IRN-150. Stir for 10 min.

Filter through paper.

Add the other additives according to Table 1.2.

1.2.1

Optional Additives

Protease inhibition

Add protease inhibitor cocktail in lysis solution

until 10 mL/mL is reached.

1% Bromophenol blue does not

go into solution without Tris

The inhibitor cocktail inhibits

most proteases during extrac-

tion; irreversible inhibition is

only obtained upon precipita-

tion of the sample.

Step 1: Sample Preparation290

Phosphatase inhibition Add Na-vanadate and NaF until 1 mmol/L

until 1 mmol/L of each is reached.

The lysis solution can be produced in larger quantities – without

DTT – and stored frozen in aliquots at a temperature deeper than

–60 C.

Tab. 1.3: Standard rehydration solution.

8 mol/L urea 4.8 g

0.5% (w/v) CHAPS 50 mg

0.28% (w/v) DTT 28 mg

10% (v/v) glycerol 1.2 mL

1.25% (v/v) Pharmalytes pH 3–10 *) 125 mL

0.002% Bromophenol blue **) 10 mL

Water, deionized, make up to 10 mL

*) or IPG buffer of respective pH interval

**) from 1% Bromophenol blue solution

Removal of isocyanate If the purity of the urea is in doubt:

Dissolve 4.8 g urea in deionized water contain-

ing 10% (v/v) glycerol, fill up to 10 mL.

Warm the tube to get the urea completely in

solution: Do not exceed 37 C.

Add 100 mg mixed bed ion exchanger Amberlite

IRN-150. Stir for 10 minutes.

Filter through paper.

Add the other additives according to Table 1.3.

The rehydration solution can be produced in larger quantities – with-

out DTT – and stored frozen in aliquots at a temperature at –20 C or

colder.

1.2.2

Liquid Samples

To analyze protein solutions such as serum, plasma etc. the solubiliz-

ing mixture is diluted to the desired protein concentration with rehy-

dration solution.

Diluted samples In general, the lower additive concentration like in

the rehydration solution should be adequate. However, some samples

might need higher concentrations, indicated on the right half of Table

1.4.

Repeated freeze thawing must

be avoided. Add DTT shortly

before use.

Repeated freeze thawing must

be avoided. Add DTT shortly

before use.

Very diluted samp les like cell

culture supernatant need to be

precipitated, usually with 100%

(w/v) TCA.