Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
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
2
�Use of enzymes (AP
and HRP) linked to
antibodies to visualise
�Ease of removal to allow
MS analysis
Oct 2011 SDMBT
Structure of stains
3Oct 2011 SDMBT
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
6
(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
7
more Ag+ reduced to Ag
Protein surrounded by a visible layer of colloidal silver
Oct 2011 SDMBT
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
8
�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
Oct 2011 SDMBT 9
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
10
(Samuel Roberts Noble Foundation)(Charrin et al, Biochem. J. 2003, 373: 409–421)
Oct 2011 SDMBT
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
11
�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
Oct 2011 SDMBT
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
Oct 2011 SDMBT 12
�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
Oct 2011 SDMBT 13
Fluorescent dye staining
Other fluorescent dyes
Deep Purple (GE Biosciences)
Krypton (Thermo Scientific)
Flamingo (BioRad)
Oct 2011 SDMBT 14
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
Oct 2011 SDMBT 15
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.
Oct 2011 SDMBT 16
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
Oct 2011 SDMBT 17
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
18Oct 2011 SDMBT
Multiplexing
- Works with fluorescent dyes and laser
excitation
Oct 2011 SDMBT 19
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
20Oct 2011 SDMBT
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
Oct 2011 SDMBT 21
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
Oct 2011 SDMBT
Comparison of staining methods
�Coomassie
staining has 20 fold
range of linearity
Intensity
(1 – 1000)
�Silver staining has
40-fold range of
linearity
23
(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