Lecture 5 Part 2 Visualisation. Oct 2011 SDMBT 1

Transcription

1 Lecture 5 Part 2 Visualisation 1

2 Staining vs Western blotting Western blot Use of antibodies to recognise protein(s) Use of enzymes (AP and HRP) linked to antibodies to visualise Staining Direct, specific interactions with protein(s) Ease of removal to allow MS analysis 2

3 Structure of stains 3

4 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 Conventional Coomassie blue staining can detect ng of protein Sensitivity still considerably less than silver staining or fluorescence staining Compatible with MS (Sigma) 4

5 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 3. Destaining typical destaining solution 25% MeOH, 8% AcOH change solutions many times until background clear. (Sigma) 5

6 Coomassie Blue Staining (Kendrick Laboratories) 6

7 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 Protein surrounded by a visible layer of colloidal silver 7

8 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 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) 8

9 Silver staining Typical staining protocol 1. Fixing Typically 40% methanol, 10% AcOH for 0.5 hr 2. Sensitisation Typically solution containing glutaraldehyde, sodium thiosulphate in ethanol/water for about 0.5 hr followed by 2-3 washes with water 3. Silver impregnation Typically 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. Stop Typically solution containing EDTA for about 10 min and then wash with water. 9

10 Silver staining (Charrin et al, Biochem. J. 2003, 373: ) (Samuel Roberts Noble Foundation) 10

11 Fluorescent dye staining Dyes are fluorescent on association with SDS on SDSprotein complexes (SYPRO Red and SYPRO Orange) So non-specific 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 11

12 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 12

13 Fluorescent dye staining 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 13

14 Fluorescent dye staining Other fluorescent dyes Deep Purple (GE Biosciences) Krypton (Thermo Scientific) Flamingo (BioRad) 14

15 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 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. 15

16 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. 2. ProQ Diamond (Molecular probes/invitrogen) Stains all phosphoproteins Fluorescent detection 16

17 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 2. Pro-Q Emerald (Molecular Probes) also rely on periodate solution to oxidise sugars dye binds to the glyco part of the protein 17

18 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 18

19 Multiplexing - Works with fluorescent dyes and laser excitation -Narrow excitation/emission profile otherwise Excitation of one dye may lead to excitation of other dye. 19

20 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 20

21 Multiplexing of fluorescent dyes CyDyes (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 21

22 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 requires special equipment for detection (Sigma) 22

23 Comparison of staining methods Intensity (1 40) (8 160) (1 1000) Protein concentration Coomassie staining has 20 fold range of linearity Silver staining has 40-fold range of linearity Fluorescent dyes can have a linearity of up to 3 orders of magnitude Maximum, Minimum, Range 23