RPN6305PL Rev E - Hebrew University of...

iR

PN

63

05

PL R

ev E 2

00

4

instructions

Warning

For research use only.

Not recom

mended or intended

for diagnosis of disease inhum

ans or animals.

Do not use internally or

externally in humans or

animals.

product code

RPN6305

RPN6306

Deep Purple TotalProtein Stain

New

Im

proved Protocol

RPN6305PL Rev E 10/14/04 9:53 AM Page 1

● 2

Handling

Storage O

n receipt, store in afreezer at -15 ºC

to -30 ºC.

Expiry The product is stable forat least 3 m

onths when

stored under therecom

mended conditions.

Deep P

urple total proteinstain is sensitive toprolonged exposure tolight. Long term

storage ofthe reagent should be inthe light tight container inw

hich it is provided.

Page finder Com

ponents 3

Safety warnings and precautions

3

Description 5

Procedure

6 A

pplications 6

Critical parameters

7

Solutions and reagents required 9

Protocol 11

1. Gel electrophoresis

11 2. Fixation

11 3. G

el staining 12

4. Blot staining

135. D

e-staining PVD

F blots14

6. De-staining nitrocellulose blots

147. Visualization

147.1. Flat-bed laser based fluorescence

imaging system

s 14

7.2. Imaging w

ith UV light sources

15 8 M

anual spot picking16

Additional information

17 R

e-staining of gels 17

Alternative staining trays

17 A

lternative imaging instrum

ents 17

Use of D

eep Purple w

ith Ettan DIG

E 18

Cleaning of im

aging instruments

18 C

leaning and preparation of Bind-Silane

coated plates 19

Troubleshooting guide 21

Quality control 24

Related products 25

References 25

Legal 26

Product information

26


● 3

Components

This pack contains thefollow

ing ●

RP

N6305

Deep P

urple Protein

Stain, 5 ml R

econstitutesto 1 litre.

●R

PN

6306 D

eep Purple P

roteinStain, 25 m

lR

econstitutes to 5 litres.

Solution supplied containsD

eep Purple total protein

stain in 50% (v/v) D

MSO

and 50% (v/v) acetonitrile.

Safety warnings and

precautions W

arning: For research use only. Not

recomm

ended or intended for diagnosis ofdisease in hum

ans or animals. D

o not useinternally or externally in hum

ans or animals.

As all chem

icals should be considered aspotentially hazardous. W

e thereforerecom

mend that this product is handled only

by those persons who have been trained in

laboratory techniques, and that it is used inaccordance w

ith the principles of goodlaboratory practice. W

ear suitable protectiveclothing such as laboratory overalls, safetyglasses and gloves. C

are should be taken toavoid contact w

ith skin or eyes. In the case ofcontact w

ith skin or eyes wash im

mediately

with w

ater. See MSD

(s) and/or SS(s) forspecific com

ponent handling instructions.

Note: T

he protocol requires the use of aceticacid and m

ethanol or ethanol, sodiumhydrogen carbonate (N

aHC

O3 ) and sodium

carbonate (Na

2 CO

3 ).

Warning:A

cetic acid causes burns and is anirritant. Please follow

the manufacturer's

safety data sheet relating to the safe handlingand use of this m

aterial.

Warning:M

ethanol is toxic and flamm

able.Please follow

the manufacturer's safety data


● 4

sheet relating to the safe handling and use ofthis m

aterial.

Warning:Sodium

carbonate (Na

2 CO

3 ) is anirritant. Please follow

the manufacturer's

safety data sheet relating to the safe handlingand use of this m

aterial.

Note: T

he protocol may involve the use of

UV

illumination devices and / or laser based

imaging instrum

ents.

Warning:In the case of both U

V illum

inationor laser scanning it should be ensured thatproper and effective safety regulations arefollow

ed. When using U

V illum

ination, a fullface U

V protective visor should be w

orn.


● 5

Description D

eep Purple™ total protein stain is a naturally occurring,

biodegradable, fluorescent compound extracted from

a fungal species(1) and it has been developed as an ultra-sensitive fluorescent stain forthe detection of proteins in-gel and blots follow

ing electrophoreticseparation (2). D

eep Purple has been shown to be up to 8 tim

es more

sensitive than similar products although, as w

ith all protein stainingm

ethods certain individual proteins can exhibit different stainingcharacteristics. D

eep Purple is an environmentally friendly stain w

hichis diluted in w

ater for use and therefore allows for easy and convenient

disposal. The stain m

ay be used in conjunction with a range of

excitation sources including UV

light boxes, broad range visible lightsources and lasers (see figure 1).

Wavelength (nm

)

Relative intensity

Figure 1. Fluorescence excitation and emission spectra of D

eep Purplestained proteins in gel plugs taken from

a 2D gel.


● 6

Procedure D

eep Purple is simple to use and reliable. G

els are run and then fixed instandard conditions using, for exam

ple, an acetic acid / methanol

solution. A staining protocol is provided for use w

ith both free-floatinggels, gels im

mobilized on glass plates and for proteins blotted onto

PVD

F or nitrocellulose mem

branes. The stain can therefore be used for

standard 1D and 2D

gel electrophoresis and for protein blots.

The stain is stable as supplied at –15 °C

to –30 ºC for at least 3 m

onthsand it has not been observed to generate particles or sedim

ent overtim

e. Working solution (1:200 dilution) is stable at 2–8 °C

for up to 1w

eek and 24 h at room tem

perature, while stained gels can be stored

for many w

eeks at 2–8 °C w

ithout significant loss in sensitivity. Inaddition, if som

e signal is lost, it is possible to re-stain gels and gainvirtually the sam

e intensity signal. Gels can also be stained using

Coom

assie post staining for manual spot picking.

The stain protocol allow

s for flexibility within individual experim

entalw

ork-flows, providing a rapid protocol of less than 3 hours for speedy

results or an overnight fix step for maxim

um sensitivity and w

ork-flowconvenience.

Applications T

he stain can be used on pre-cast or lab-cast, Bis-T

ris, Tris-A

cetate andT

ris-Glycine gels; w

ith gel systems containing low

or high SDS levels;

and is compatible w

ith other buffer systems such as M

ES, M

OPS or

tricine. Deep Purple is also suitable for protein blot staining using

PVD

F or nitrocellulose mem

branes.

Deep Purple stained gels can be im

aged on a variety of instruments.

Flat bed laser based fluorescence scanners are strongly recomm

endedfor optim

al imaging of D

eep Purple stained gels, for example using the

Typhoon™ scanner. H

owever the stain can also be visualized w

ith longw

avelength UV

illumination using - 365 nm

or blacklight blue UV

Asources and im

ages can be captured on a suitable CC

D or video cam

era


● 7

system.

Deep Purple fits into the standard 2D

gel electrophoresis work-flow

and is particularly suitable for use with the E

ttan™ D

IGE

system. T

herecom

mended w

ork-flow for this system

involves the matching of D

eepPurple stained preparatory gels w

ith CyD

ye™ labeled analytical gels.

Deep Purple has been show

n to be compatible w

ith DeC

yder™difference analysis softw

are and the stain is compatible w

ith manual or

automated spot picking and m

ass spectrometry for protein

identification applications.

Critical parameters

●R

ead the entire protocol thoroughly before using the kit.

●E

nsure that the containers used for gels are clean and do not containany contam

inants. A w

ide variety of non-metallic containers can be

used with this stain, including polypropylene, polystyrene or Pyrex™

glass (for details see ‘Additional Inform

ation’, page 17).

●It is essential to ensure that plates to be coated w

ith Bind-Silane are

prepared to the highest standard (see ‘Additional Inform

ation’, page17).

●It is recom

mended to use gloves that are not pow

dered. Wash new

gloves prior to handling plates, containers or gels. Any pow

dertransferred to the gel m

ay show up as speckles on im

ages.

●D

uring preparation of plates for gel casting, it is advised to employ

methods that m

inimize generation of dust particles. T

he use of anytype of paper tow

el will generate particulate m

atter that will be

visualized as ‘speckles’. Plates should be cleaned using lint free cloths,such as C

rew™

Wipers.

●For gel staining, during the protein staining step a volum

e ofw

orking stain solution equivalent to at least a 10-fold excess of thegel volum

e should be used. For blot staining a volume of w

orking


● 8 solution sufficient to cover the m

embrane to a m

inimum

depth of 1.5cm

is recomm

ended to use.

During all other steps a volum

e equivalent to ~20-fold excess of the gel

volume should be used (see Table 1, below

).

Table 1.Typical stain and gel processing solution volum

es for the Deep

Purple total protein stain protocol.

Electrophoresis

Gel D

imensions

Stain Volum

eProcessing Solution

System(cm

)(m

l)V

olume (m

l)

Hoefer™

miniV

E10 x 10 x 0.05

50100

Hoefer SE

26010 x 10 x 0.05

50100

Hoefer SE

40018 x 16 x 0.1

250500

Hoefer SE

60018 x 16 x 0.1

250500

Hoefer SE

66018 x 24 x 0.1

5001000

Ettan D

ALT

six20 x 26 x 0.1

5001000

Ettan D

ALT

twelve

20 x 26 x 0.1500

1000

●D

o not dilute the stain beyond 1:200 as this will result in reduced

intensity and sensitivity.

●D

o not re-use the stain solution as this may result in a significant loss

of sensitivity.

●D

uring the process gel containers should be covered to exclude lightand agitated gently on a m

ixer platform.


● 9

Solutions and reagents required

Amersham

Biosciences reagents SD

S : PlusOne™

(code number 17-1313-01), U

SB reagent (U

S75819-100g or U

S85819-1kg) or similar high quality m

aterial (such as BD

HSpecially Pure grade).

Acrylam

ide gel and other related electrophoresis reagents are availablein the PlusO

ne range and are detailed in the current BioD

irectory™catalogue.

Additional reagents required Sodium

hydrogen carbonate (NaH

CO

3 ) and sodium carbonate

(Na

2 CO

3 ).

High purity w

ater (double distilled, RO

or equivalent).

Acetic acid, glacial.

Methanol/ethanol.

Note:H

igh purity water (R

O quality or better) should be used as a

diluent for Deep Purple total protein stain and for preparing all gel

processing solutions.

Fixation solution for gels7.5%

(v/v) acetic acid / 10% (v/v) m

ethanol or ethanol.

Add 75 m

l acetic acid and 100 ml m

ethanol to 825 ml w

ater.

Wash solution for large and backed gels

35 mM

sodium hydrogen carbonate

(NaH

CO

3 ) and 300 mM

sodiumcarbonate (N

a2 C

O3 ) in w

ater.

This can be achieved by dissolving 2.94 g of sodium

hydrogencarbonate (N

aHC

O3 ) and 31.8 g of sodium

hydrogen carbonate in 750m

l of water and adding w

ater to a final volume of 1000 m

l. The pH

of


● 1

0

the solution should be pH 10-11 and should be verified. T

his solutioncan be stored for up to 2 w

eeks.

Wash solution for free floating gels and blots

200 mM

sodium carbonate (N

a2 C

O3 ) in w

ater.

This can be achieved by dissolving 21.2 g of sodium

carbonate(N

a2 C

O3 ) in 750 m

l of water and adding w

ater to a final volume of

1000 ml. T

he pH of the solution should be at least 11 and should be

verified. This solution can be stored for up to 2 w

eeks.

Working stain solution for gels and blots

1 in 200 dilution of Deep Purple in w

ater.

For the appropriate volume to use refer to Table 1 (see page 8). T

hissolution should be m

ade fresh at the time of use by adding an

appropriate aliquot of Deep Purple to w

ater in the gel staining tank. Ifnecessary it is possible to store this solution, protected from

exposureto light, for up to 1 w

eek at 2-8 °C or 24h at room

temperature.

Stabilization solution for gels7.5%

(v/v) acetic acid.

Make a 7.5%

acetic acid solution by adding 75 ml glacial acetic acid to

925 ml w

ater.

Methanol/Acetic acid solution for w

ashing of PVDF blots

10% (v/v) m

ethanol + 7.5% acetic acid.

Make a 10%

methanol / 7.5%

acetic acid solution by adding 75 ml of

glacial acetic and 100 ml of m

ethanol to 825 ml w

ater.


● 1

1

Protocol

❶ Gel electrophoresis

1.1.Perform electrophoresis according to established techniques.

Note: If visual orientation is required on 1D

gels, Rainbow

™ M

arkers(R

PN800) m

ay be used. If a tracking dye is used, such as bromophenol

blue in the loading buffer, it is recomm

ended to run the dye front justoff the bottom

of the gel. For blotting it is particularly important that

the tracking dye and ion front are run off the base of the gel as ionscan interfere w

ith protein binding to the mem

brane.

1.2. If blotting, perform electro-transfer according to established

techniques and proceed to step 4.

❷ Fixation

2.1.Place an appropriate volume of 7.5%

(v/v) acetic acid / 10%(v/v)

methanol into the containers that w

ill be used to process gels. The

recomm

ended volume of fixation solution required is ~20 fold excess of

the gel volume (see Table 1, page 8).

Note:A

lternative fixation solutions that have been used successfullyw

ith Deep Purple total protein stain are:

●7.5%

acetic acid / 10% ethanol

●7.0%

acetic acid / 30% ethanol

2.2.Dism

antle the electrophoresis apparatus.

●For free floating gels rem

ove the gel from the plates by floating the

gel off with gentle agitation in the fix solution.

●For backed gels place the gel and plate directly into the fix solution.

Note:Place only one gel in each container. T

he stacking gel can be left


● 1

2

attached to help with gel orientation.

2.3.Incubate in the fixation solution, for a minim

um of 1 hour, at

room tem

perature with gentle agitation.

Note:O

vernight fixation should be used for backed gels, large format

gels and thick gels (≥1.5 m

m) and it is also recom

mended for

applications where m

aximum

sensitivity is required.

❸Gel Staining

3.1.Take the stain out of the -15 °C to -30 °C

freezer and allow to

stand at room tem

perature for 5–10 minutes.

3.2.Pour off the fixation solution and replace with the w

ash solutionin ~20 fold excess (See Table 1, page 8 for all volum

es). Wash w

ithgentle agitation for 30 m

inutes.

Note: For backed gels and thick gels, the w

ash solution should be 35 m

M sodium

hydrogen carbonate (NaH

CO

3 ) and 300 mM

sodiumcarbonate (N

a2 C

O3 ) and for free floating gels the w

ash solution shouldbe 200 m

M sodium

carbonate (Na

2 CO

3 ).

3.3.Pour off the wash solution and replace w

ith water (10 fold excess

of the gel volume). B

riefly shake the stain concentrate and add Deep

Purple to make a 1:200 dilution. C

over the container to create a darkenvironm

ent and incubate for 1 hour at room tem

perature with gentle

agitation.

Note:T

he solution is light sensitive and should be kept out of brightlight.

Note:C

ontainers can be wrapped in foil or covered w

ith black plastic.It is not necessary to elim

inate light completely, only to ensure that

bright light is significantly reduced. Alternatively, containers, w

ith lids,that are a solid colored plastic m

ay be used.

3.4.Pour off the stain and replace with 7.5%

(v/v) acetic acid. Cover


● 1

3

the container to create a dark environment and incubate w

ith gentleagitation for at least 15 m

inutes. Repeat the acetic acid step once. T

hegel can be im

aged at this stage.

Note:

If speed is more im

portant than background levels, the gel canbe im

aged after one acetic acid step. Further washes in acetic acid can

be performed to reduce the background still further if necessary. A

fterim

aging, the gels can be stored in the dark in 7.5%(v/v) acetic acid at

2–8 ºC for several w

eeks. This allow

s for further imaging at a later date

if required.

➍Blot Staining

4.1.Take the Deep Purple out of the freezer and allow

to stand atroom

temperature for 5-10 m

ins.4.2. Follow

ing electro-transfer, place the wet m

embrane in w

ater andw

ash for 5 mins.

4.3. Pour off water and replace w

ith 200 mM

sodium carbonate

(Na

2 CO

3 ) and wash for 5 m

ins.4.4.Pour off the 200 m

M sodium

carbonate (Na

2 CO

3 ) solution andreplace w

ith water. A

dd Deep Purple to m

ake a 1:200 dilution andstain the blot for 15 m

ins. Avoid placing the blot in direct light,

although it is not necessary to eliminate light com

pletely. N

ote:For small blots add 250 µL

Deep Purple‘ to 50 m

L w

ater. Forlarge blots add 2 m

L of D

eep Purple‘ to 400 mL

of water.

4.5. If using PVD

F mem

branes go to Step 5. For nitrocellulosem

embranes proceed to Step 6.


● 1

4

➎De-staining PVDF blots

5.1.Pour off the staining solution and replace with 10%

methanol /

7.5% acetic acid and w

ash the blot for 5 mins.

Note: T

his will cause the blot to appear green.

5.2.Rinse the blot in 100%

methanol for 2-3 m

ins until greenbackground on the blot has been com

pletely removed.

5.3.Dry the m

embrane. T

he blot is ready for imaging or further

analysis.

➏De-staining nitrocellulose blots

6.1.Pour off the staining solution and replace with 200 m

M sodium

carbonate (Na

2 CO

3 ) solution and wash for 5 m

ins.6.2.R

emove the 200 m

M sodium

carbonate (Na

2 CO

3 ) solution,replace w

ith water and w

ash for 5 mins.

6.3.Repeat the w

ater wash step.

6.4.Dry the m

embrane. T

he blot is ready for imaging or further

analysis.

➐Visualization of gels and blots

7.1

Flat-bed laser based fluorescence imaging system

s7.1.1.E

nsure that the scanning bed of the laser is clean and free fromsm

ears and particles. Follow recom

mended procedures provided w

iththe instrum

ent.

Note: O

n the Typhoon scanner it has been shown that fluorescent

contamination on the platen can be elim

inated by wiping the surface

with 10%

(v/v) H2 0

2(hydrogen peroxide) follow

ed by a rinse with

double distilled water (see ‘A

dditional Information’, page 18 for full

details).

7.1.2.Set up the scanner as recomm

ended in the relevant systemoperational m

anual.


● 1

5

For example, the follow

ing settings are recomm

ended for use with a

Typhoon scanner;

Excitation : G

reen laser (532 nm)

Em

ission: 560LP or 610B

P filter.

Pre-scan using 1000 micron resolution and then scan using a 100

micron resolution.

Note:If the pre-scan show

s saturated bands/spots, reduce the PMT

voltage rating and pre-scan again. If the pre-scan shows too low

signalincrease the PM

T voltage rating and pre-scan again. D

eep Purple canalso be im

aged on a Typhoon scanner using the blue laser (457 nm or

488 nm). If using an alternate fluorescent scanner, for the best optim

alim

ages, scan using as similar settings as possible to those

recomm

ended.

7.1.3.Process the image according to experim

ental requirements and

the instructions for the relevant software program

.

7.2

Imaging w

ith UV light sources

7.2.1.Place the gel onto the UV

transilluminator or (blacklight blue

365 nm w

avelength emission is recom

mended) and follow

theoperating and safety instructions as relevant for the excitationinstrum

ent and imaging system

. Images can be captured using

appropriate camera system

s and filters (film, video, C

CD

).

Note: For long periods of illum

ination it is advisable to place the gelon a glass plate, raised on spacers above the transillum

inator, in orderto reduce heat dam

age to the stained proteins. Cooling the gel prior to

visualization can also help reduce fading.


● 1

6

➑M

anual spot/band picking8.1.C

olorimetric post-staining using C

oomassie™

Brilliant B

lue CB

Bor silver.

If desired, gels can be re-stained with either C

BB

or silver as describedin the application note 11-0008-18 A

A, 2004-06. Spots can then be

cut manually.

8.2.Picking spots using UV

A or B

-illumination.

If manually picking spots using a U

VA

-transillumination, it is advisable

to place the gels onto a glass plate. Prolonged exposure to a strong UV

source will degrade the D

eep Purple signal, with a half-life in the region

of 15 to 30 minutes. B

lack light blue UV

A lam

ps are recomm

ended fortransillum

inators as they are more suited to the spectral profile of

epicocconone and produce lower background light.


● 1

7

Additional information

Re-staining of gels G

els that have lost sensitivity over time or through incorrect storage

can be re-stained. In addition gels can be photo-bleached by prolongedexposure to U

V light and then re-stained. In all cases the m

ain protocolshould be follow

ed starting at the ‘Staining’ process (step 3, page 12).

Alternative staining trays Staining gels w

ith Deep Purple in stainless steel trays and the Processor

Plus gives comparable results. T

he limitation for large gels in Processor

Plus is that the maxim

um volum

e for the 29 x 35 cm tray is 400 m

l, sow

e recomm

end that the Processor Plus should be programm

ed with the

double number of steps. T

he use of Processor Plus gives higher andlow

er signal intensity for small and large gels, respectively. T

he use ofstainless steel trays as an alternative to the plastic trays results in tw

o-fold less sensitivity in a dilution series on a SD

S-PAG

E gel and few

erspots in 2-D

gel patterns.

Alternative imaging instrum

entsIm

aging is best performed using laser scanning fluorescence flat bed

imaging system

s, such as the Typhoon imager. T

his instrument can be

used to excite Deep Purple at 457 nm

, 488 nm or 532 nm

with

emission being collected through a 560 nm

long pass filter or a 610 nmband pass filter. W

hen using alternative laser based fluorescencescanners select laser w

avelength and emission filters that are closest to

those presented in the main protocol. O

ptimization of the scanning

process may be required to account for the relative pow

er of differentlasers and the use of alternative filter settings.

Many m

akes of UV

transilluminator are available that produce light of

UV

A (eg 365 nm

wavelength). B

lacklight blue UV

A sources are

particularly compatible w

ith gels stained with D

eep Purple.


● 1

8

Gels excited by U

V light can be visualized in a num

ber of ways but if

further image analysis is required it is recom

mended that the im

age iscaptured on a C

CD

camera system

, such as the BioC

hemi™

Darkroom

(UV

P); the ethidium brom

ide (610 nm band pass) em

ission filter hasbeen used to give satisfactory im

ages from D

eep Purple stained gels.A

n alternative is imagers based on black light blue light sources. T

heseare sufficiently close to the excitation peak of D

eep Purple to produceuseful fluorescence.

Use of Deep Purple w

ith Ettan DIGE D

eep Purple fits into the standard 2D gel electrophoresis w

ork-flowand is particularly suitable for use w

ith the Ettan D

IGE

system. T

herecom

mended w

ork-flow involves the m

atching of Deep Purple post-

stained preparatory gels with C

yDye pre-labeled analytical gels.

How

ever, if it is necessary to post-stain analytical gels with D

eepPurple, it is recom

mended to im

age the protein stain using 457 nmlaser excitation in conjunction w

ith a 610 nm band pass em

ission filter(or equivalent if not using a Typhoon scanner). T

his will m

inimize any

potential cross-talk between D

eep Purple and the CyD

ye DIG

E fluors.

Deep Purple has been show

n to be compatible w

ith DeC

yder differenceanalysis softw

are and the stain is compatible w

ith manual or

automated spot picking and m

ass spectrometry for protein

identification applications.

Cleaning of imaging instrum

ents D

eep Purple total protein stain may leave a fluorescent residue on the

scanner platen. If the platen is not thoroughly cleaned, this residue caninterfere w

ith subsequent scans producing high background levels.

The follow

ing cleaning procedure has been shown to be com

patiblew

ith the Typhoon fluorescent imager to rem

ove contamination caused

by fluorescent products. Com

patibility of this procedure with

alternative instruments w

ould need to be investigated.


● 1

9

●W

ipe the platen with 10%

H2 0

2(hydrogen peroxide).

●R

inse the platen with high purity w

ater.

●T

his operation should be carried out using lint free cloths, such asC

rew W

ipers. A pre-scan can be done to check for contam

inants thatm

ay affect results of scans.

●If the scanner is shared and used for scanning dyes/stains other than

Deep Purple, it is suggested that 1D

or free floating gels are placedonto a clean glass plate for scanning purposes. T

his will reduce the

possibility of cross contamination. If this m

ethod is used, the scanner‘Focal plane’ setting m

ay need to be set to +3mm

instead of platen (ifpossible).

Cleaning and preparation of Bind-Silane coated plates For com

plete removal of old gel and B

ind-Silane:

●T

horoughly scrape off any residual bound gel with a plastic scraper.

●W

ash the plates in freshly prepared 1%(v/v) D

econ™ (branded

Contrad™

in the USA

) and wash w

ith a soft sponge or brush tofurther rem

ove the gel.

●L

eave the plate to soak in 1%(v/v) D

econ overnight.

●T

he following day, w

ash the plate with a soft sponge.

●R

inse the plate with w

ater and leave the plate to soak in 1%(v/v)

HC

l for 1 hour.

●W

ash the plate in 1%(v/v) D

econ with a soft sponge or brush, then

rinse with high purity w

ater and leave to dry.

●B

efore using the plate, wipe over w

ith ethanol, using a lint free clothsuch as C

rew W

ipers.

●B

ind-Silane should be applied to plates using a lint free cloth so thatthe solution is spread evenly. L

ong strokes, from one edge of the plate

to the other, should be used, until the plate looks dry. The plate


● 2

0

should then be covered with a dry cloth to prevent particulate

contamination and left for at least 2 hours to dry com

pletely.


● 2

1

❶Overall low signal intensity

Ensure that all steps in the protocol have

been included and that incubation times

and wash volum

es have been followed

correctly.

Ensure that the stain w

as not exposed tobright light either as the w

orking stocksolution or particularly during the stainingprocess.

Ensure that the sodium

hydrogen carbonate(N

aHC

O3 ) and sodium

carbonate(N

a2 C

O3 ) solution has correct pH

.

Perform the 1:200 dilution of the D

eepPurple stock solution only w

hen required.

Re-use of the stain is not recom

mended.

Ensure D

eep Purple dilution was 1:200 in

water and that the correct volum

e was used

for the volume of the gel (also taking into

account the size of the container).

Check that the gel w

as incubated in thestain for at least the recom

mended period

of time (staining for longer periods has not

been seen to increase background).

Re-scan the gel at a higher PM

T voltage

(applicable to laser based fluorescentscanning instrum

ents only).

Use a longer exposure tim

e (for CC

D based

imaging instrum

ents).

Troubleshooting guideProblem

sPossible causes and solutions


● 2

2

❷

Overall elevatedbackground

❸Localized patches of highbackground signal

Fading of stain in a stored gel – ensure gelsare stored in the recom

mended conditions

and protected from light. R

e-stain the gel ifnecessary.

Ensure correct fixation solution w

as used.

Prolong fixation to an overnight step.



aHC

O3 ) and sodium

carbonate(N

a2 C


.

Perform extra w

ashes with 7.5%

(v/v) aceticacid after the staining step.

Ensure correct volum

es of all solutions havebeen used for the volum

e of gel (also takinginto account the size of the container).

Ensure that the platen of the im

agingsystem

is not contaminated w

ith fluorescentcom

pounds.

When using thicker gels or backed gels use

an overnight fixation step.

Use a high quality SD

S in the preparationand running of the gel (such as the PlusO

necode num

ber 17-1313-01 or USB

productU

S75819); replace pre-made running

buffers if necessary.

Platen of the imaging system

may be

contaminated w

ith fluorescent compounds

– follow the recom

mended cleaning

procedure.


● 2

3

❹Spots and streaks clearlyvisible in the background

❺Small ‘speckles’ are seen

on the image

The SD

S front on gels will be stained by

Deep Purple and w

ill appear as a dark bandacross the gel. T

racker dyes, such asbrom

ophenol blue, can absorb fluorescentlight resulting in a band that appearsclearer than the background. To avoid thistracker dyes should be run off the bottomof the gel.

Handle gels w

ith care as physical damage

to gels may give a background stain in that

area. Use clean pow

der free gloves.

If a dark stained area is seen at low m

ass /high pI on a 2D

gel ensure that the fixationsolution is as recom

mended and the

fixation step is performed overnight.

If using backed gels ensure that the gelplates are properly cleaned before use andthat all gel m

aterial from previous

experiments is com

pletely removed.

Ensure that B

ind-Silane coated plates areevenly coated, dried efficiently and freefrom

particulate contamination.

Platen of the imaging system

may be

contaminated w

ith fluorescent compounds

– follow the recom

mended cleaning

procedure.

Use clean pow

der free gloves.

Ensure gel containers are perfectly clean

and rinsed before use. Ensure containers are


● 2

4

❻Boundary or negativestaining effects (bands /spots surrounded by alighter stained area)

➐Swelling of gels

free of contaminating fluorescent

compounds.



aHC

O3 ) and sodium

carbonate(N

a2 C


andvolum

e.

Inefficient removal of buffer com

ponentsfrom

the gel during fixation. Use m

ethanolor ethanol in the fixation solution andperform

fixation overnight.

Use a high quality SD

S in the preparationand running of the gel (such as the PlusO

necode num

ber 17-1313-01 or USB

productU

S75819); replace pre-made running

buffers if necessary.

Swelling of free floating gels can be reduced

by adding 10% (v/v) m

ethanol in the finalacetic acid steps and the stabilizationsolution.

Quality control E

ach batch of Deep Purple undergoes rigorous quality control to ensure

optimum

and consistent performance.


● 2

5

Related products A

mersham

Biosciences offers a com

prehensive range of electrophoresisreagents and hardw

are all with proven com

patibility to ensurereproducible high quality results. For a com

plete listing of productsavailable see the current A

mersham

Biosciences catalogue or visit our

web site at w

ww

.amersham

.com.

References

1. Bell, P.J.L

. and Karuso, P., J. A

m. C

hem. Soc.,125, 9304-5 (2003).

2. Mackintosh, J.A

. et.al., Proteomics,3,2273-88 (2003).


● 2

6

LegalG

E and G

E M

onogram are

trademarks of G

eneral Electric

Com

pany.

Am

ersham and A

mersham

Biosciences are tradem

arks of

Am

ersham plc

BioD

irectory, CyD

ye, DeC

yder,

Deep P

urple, Ettan, H

oefer,

PlusO

ne, Rainbow

and Typhoon

are trademarks of A

mersham

Biosciences Lim

ited.

Crew

is a trademark of

Kim

berly-Clark.

Decon and C

ontrad are

trademarks of D

econ

Laboratories.

Pyrex is a tradem

ark of Corning.

BioC

hemi is a tradem

ark of

UVP

.

Coom

assie is a trademark of IC

I

Am

ericas, Inc.

All goods and services are sold

subject to the terms and

conditions of sale of the

company w

ithin the Am

ersham

Biosciences G

roup which

supplies them. A

copy of these

terms and conditions is

available on request.

© A

mersham

Biosciences U

K

Limited 2

00

4 - A

ll rights

reserved

Deep P

urple total proteinstain

RP

N6

30

5R

PN

63

06

● 2

6

Product information

ProductCode

http://ww

w.am

ersham.com

Amersham

Biosciences UK

Limited

Am

ersham P

lace Little Chalfont B

uckinghamshire H

P7 9N

A U

K

Amersham

Biosciences AB

SE-751 84 Uppsala Sw

eden

Amersham

Biosciences Corp

800 Centennial Avenue P

O B

ox 1327 Piscataw

ay NJ 08855 U

SA

Amersham

Biosciences Europe GmbH

Munzinger Strasse 9 D

-79111 Freiburg Germ

any

Deep P

urple total protein stain is exclusively licensedto A

mersham

Biosciences from

Fluorotechnics Pty

Ltd.

Deep P

urple total protein stain may only be used for

applications in life science research.

“CyD

ye: this product or portions thereof ism

anufactured under license from C

arnegie Mellon

University under patent num

ber 52

68

48

6 and other

patents pending. Som

e of these products may only be

available to collaborators and customers w

ithincertain of our technology access program

mes. The

purchase of CyD

ye DIG

EFluors includes a lim

itedlicense to use the C

yDye Fluors for internal research

and development, but not for any com

mercial

purposes. A license to use the C

yDye Fluors for

comm

ercial purposes is subject to a separate licenseagreem

ent with A

mersham

Biosciences’.


Deep Purple Total Protein Stain RPN6305/RPN6306

•. Solutions used in every step should be 20 fold the gel volume, except the staining solution that shouldbe 10 fold the gel volume.

•. Refer to the protocol booklet for a full and detailed explanation of the steps summarised below.

FIXFix gels overnight in 7.5% acetic acid, 10% methanol/ethanol.

WASHLarge, backed and thick gels: Wash for 30 minutes in 300 mM sodium carbonate (Na2CO3), 35 mMsodium hydrogen carbonate NaHCO3 (this solution should have a pH of 10 -11). Small and free-floating gels: Wash for 30 minutes in 200 mM sodium carbonate (Na2CO3) (this solutionshould have a pH of at least 11).

STAINStain for 1 hour at room temperature covered from light using a 1:200 dilution of the Deep Purpleconcentrate. Ensure that the Deep Purple concentrate has equilibrated to room temperature prior to dilutingwith pure water.

STABILISEWash in 7.5% acetic acid for 2 x 15 minutes covered from light. Further washes in this solution can beperformed if desired.

IMAGETyphoon: 532 nm (excitation) and 610 nm BP30 or 560 nm LP (emission). Pre-scan at 1000 µm


RPN6305PC Rev E 2004

resolution to determine optimal PMT settings then scan at 100 µm resolution with optimal PMT. Otherimaging advices: For more details see Additional information in protocol booklet.

STORAGEAfter imaging, the gels can be stored in the dark in 7.5% acetic acid at 2-8 degrees for several weeks. Thisallows for further imaging at a later date if required.

Deep Purple is a trademark of Amersham Biosciences Limited.

Amersham and Amersham Biosciences are trademarks of Amersham plc.

GE and GE Monogram are trademarks of General Electric CompanyAll goods and services are sold subject to the terms and conditions of sale of the company within the AmershamBiosciences Group which supplies them. A copy of these terms and conditions is available on request

© Amersham Biosciences UK Limited 2004 - All rights reserved

WarningFor research use only.Not recommended or intended for diagnosis of disease inhumans or animals. Do not use internally or externally in humans or animals.

http://www.amersham.com

Amersham Biosciences UKLimited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK

Amersham Biosciences AB SE-751 84 Uppsala Sweden

Amersham Biosciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA

Amersham Biosciences Europe GmbH Munzinger Strasse 9 D-79111 Freiburg Germany


Date post:	27-Apr-2018
Category:	Documents
Upload:	ngophuc
View:	216 times
Download:	2 times

RPN6305PL Rev E - Hebrew University of...

Documents