Lecture 5 Part 2

Visualisation

Oct 2011 SDMBT 1

Staining vs Western blotting

Western blot

�Use of antibodies to

recognise protein(s)

�Use of enzymes (AP

Staining

�Direct, specific interactions

with protein(s)

�Ease of removal to allow

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�Use of enzymes (AP

and HRP) linked to

antibodies to visualise

�Ease of removal to allow

MS analysis

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Structure of stains

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Coomassie Blue Staining

�Based on the binding of the Coomassie Blue

dyes (G250, R250, R350) to all proteins

Non-specific

Roughly stoichiometric – intensity roughly

propotional to concentration

4(Sigma)

�Conventional Coomassie blue staining can

detect 30-100ng of protein

�Sensitivity still considerably less than silver

staining or fluorescence staining

�Compatible with MSOct 2011 SDMBT

Coomassie Blue Staining

Typical staining protocol

1. Fixing (for SDS-PAGE optional)

Typically fixing solution 40% MeOH, 10% AcOH for 0.5 hr

For IEF gels, must fix with 20% trichloroacetic acid for 20 min to 0.5 hr

2. Staining

with Coomassie Blue solution (MeOH solution) – few min to few hours

5(Sigma)

Oct 2011 SDMBT

with Coomassie Blue solution (MeOH solution) – few min to few hours

3. Destaining

typical destaining solution 25% MeOH, 8% AcOH – change solutions

many times until background clear.

Coomassie Blue Staining

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(Kendrick Laboratories)

Oct 2011 SDMBT

Silver staining

Proteins (amine groups) react with one end of

glutaraldehyde – sensitisation

Gel impregnated with Ag+ which cluster around proteins

Ag+ is reduced by formaldehyde to Ag - autocatalytic

Other aldehyde group of glutaraldehyde oxidised by Ag+

more Ag+ reduced to Ag

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more Ag+ reduced to Ag

Protein surrounded by a visible layer of colloidal silver

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Silver staining

�Not an end-point method. The amount of development

time needs to be fixed:

•Too short = loss in sensitivity

•Too long = over-staining

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�Relationship between silver and protein not linear

�Can silver-stain after Coomassie staining

�Silver staining procedure modified to make it compatible

with MS analysis –sensitise with sodium thiosulphate only –

no glutaraldehyde (crosslinking – MS incompatible)

Oct 2011 SDMBT

Silver staining

Typical staining protocol

1. FixingTypically 40% methanol, 10% AcOH for 0.5 hr

2. SensitisationTypically solution containing glutaraldehyde, sodium thiosulphate in ethanol/water

for about 0.5 hr followed by 2-3 washes with water

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3. Silver impregnationTypically solution containing formaldehyde and silver nitrate for 20 min followed

by 2-3 washes with water

4. Development

Typically solution containing sodium carbonate and formaldehyde for about 5 min or

until satisfactory results are obtained.

5. StopTypically solution containing EDTA for about 10 min and then wash with water.

Silver staining

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(Samuel Roberts Noble Foundation)(Charrin et al, Biochem. J. 2003, 373: 409–421)

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Fluorescent dye staining

�Dyes are fluorescent on association with SDS on SDS-

protein complexes (SYPRO Red and SYPRO Orange)

�So non-specific

�Requires the use of a laser scanner/imaging system

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�Requires the use of a laser scanner/imaging system

�Can detect 2-10ng of protein – same as silver staining

�Can do Western Blot and MS after fluorescent stains

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Fluorescent dye staining

Fluorescent dyes absorb light of UV frequencies to emit light of longer wavelengths

e.g. Sypro Orange – left and Sypro Red - right

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�Newer fluorescent dyes are metal-chelate dyes that interact with the proteins

(SYPRO Rose (used for membranes) and SYPRO Ruby)

�Sypro Ruby binds to basic amino acids and the polypeptide backbone

Fluorescent dye staining

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Fluorescent dye staining

Other fluorescent dyes

Deep Purple (GE Biosciences)

Krypton (Thermo Scientific)

Flamingo (BioRad)

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Fluorescent Dye staining

Typical staining protocol

1. (native PAGE gels) need to incubate in SDS. Do not need fixing bec may

remove

the SDS coating around proteins.

2. Staining

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2. Staining

Typically a dilute solution of dye in AcOH for 30 min (depends on thickness of

gel) in the dark or wrap gel staining container in aluminium. Wash once with

AcOH solution (too long will reduce signal)

3. Detection with laser

Sypro Ruby needs fixing and staining is done overnight or with heating.

Stains for PTM proteins

Phosphorylated proteins

1. Gel Code (Pierce)

Phosphate-protein bond hydrolysed with 0.5N NaOH in the presence of

calcium ions to precipitate calcium phosphate. This reacts with ammonium

molybdate to form a complex which can be stained with methyl green.

Colorimetric – i.e. can be detected by eye.

Phosphotyrosine cannot be detected.

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Phosphotyrosine cannot be detected.

2. ProQ Diamond (Molecular probes/Invitrogen)

Stains all phosphoproteins

Fluorescent detection

Stains for PTM proteins

Glycoproteins

1. Periodic Acid-Schiff Reagent Method -

Gel is treated with a periodate solution to oxidize cis-diol sugar groups

glycoproteins. The resulting aldehyde groups react with Schiff reagent to produces

purple bands.

Colorimetric method

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2. Pro-Q Emerald (Molecular Probes)

also rely on periodate solution to oxidise sugars

dye binds to the glyco part of the protein

Fluorescent dye staining – Multiplex staining

-Multiplex staining – stain with 2 or more fluorescent dyes

-Each dye binds to a different type of protein

-Each dye excited with different wavelength UV light

-Emissions collected separately

-Advantage – one gel many results – good for comparisons

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Multiplexing

- Works with fluorescent dyes and laser

excitation

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excitation

-Narrow excitation/emission profile otherwise

Excitation of one dye may lead to excitation

of other dye.

Multiplexing of fluorescent dyes

CyDyes (GE Biosciences)

CyDyes – three dyes Cy2, Cy3 and C5

CyDye reacts with lysines of proteins (only 3% of proteins are actually labelled so

does not affect MS identification)

CyDye has a positive charge – lysine also positive – so pI unchanged

Mw of dye about 500 – so Mw of labelled protein does not change greatly from

unlabelled

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unlabelled

Multiplexing of fluorescent dyesCyDyes (GE Biosciences)

Samples are reacted with dyes before separating on gels

Each sample is labelled separatedly

Labelled samples run on same gel (so up to 3 samples run on 1 gel)

Reduces poor reproducibility if 3 gels were run for 3 samples

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Comparison of staining methods

�Coomassie staining is well established but lacks

sensitivity

�Silver staining has better sensitivity but poor dynamic

range, and contains many steps.

�Fluorescent staining has good sensitivity and dynamic

range and allows for multiplexing but is very expensive and

22(Sigma)

range and allows for multiplexing but is very expensive and

requires special equipment for detection

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Comparison of staining methods

�Coomassie

staining has 20 fold

range of linearity

Intensity

(1 – 1000)

�Silver staining has

40-fold range of

linearity

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(8 – 160)

Protein concentration

�Fluorescent dyes

can have a linearity

of up to 3 orders of

magnitude

(1 – 40)linearity

Maximum, Minimum, Range

Oct 2011 SDMBT