Chapter 7 2D PAGE Flashcards
Objectives
Describe the principles of 2D-PAGE
Describe typical staining methods used in 2D-PAGE
Describe how Western blots are used to visualise SDS-PAGE and 2D-PAGE results
Describe how a 2-D gel is analyzed through spot analysis
Things to do in workflow in proteomic analysis
Sample preparation between sample and protein mixture
Sample preparation and visualization, comparative analysis, digestion between chromatography and peptides
Mass spectrometry to get ms data
Database search for protein identification
6 staining methods for 2-PAGE analysis
Coomassie blue
Silver staining
Fluorescent
Ponceau S
Zinc Imidazole
Epitope tag
Coomassie blue dye introduction
Based on the binding of the Coomassie brillant blue dye (G-250 and R-250) to proteins
Principle of Coomassie blue dye staining
Proteins containing basic or aromatic amino acid side chains bind by hydrophobic or Van der Waal’s interactions with Coomassie Brilliant Blue dye.
This causes a spectral shift from red/brown form of the dye (A465) to the blue form of the dye (A595).
Conventional Commasie blue dye
Able to detect 30-100ng of proteins
Comparison of commasie blue staining compared to other
Sensitivity considerably LESS than silver staining or fluorescence staining
R-250 is 5-10 times more sensitive than G-250 but G-250 staining protocol is shorter
Has a linear staining response
How to increase Coomassie blue staining sensitivity
Sensitivity can be increased by using collodial Coomassie blue staining
G-250 can be mixed with methanol, phosphoric acid and ammonium sulfate to form a colloidal mix
- Can stain proteins in polyacrylamide gels without staining the gels
- Able to detect 8-10 ng of protein
Comparing CCB G-250 and CCB R-250
CCB G-250 is blue
CCB R-250 is pink
Conventional CCB effect
Staining time: 1 hour, no bands visible before de- staining
After de-staining: 1 hour in methanol, acetic acid
Colloidal CCB effect
Staining time: 1 hour bands visible in the staining tray
After de-staining, 1 hour water wash enhancement
Sliver stain introduction
Most sensitive colorimetric method for protein detection
Sliver ions interact strongly with carboxylic acid groups (Asp and Glu), imidazole (His), sulfhydryls (Cys) and amine (Lys), bound sliver able to be visualized after precipitation via reduction using reagents such as formaldehyde
Able to detect 1ng of protein
3 limitations of sliver stain
- 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
- Narrow linear dynamic range - Not compatible with analysis by Mass Spectrometry
- Formaldehyde can cross-link proteins to gel matrix
Sliver stain
Loss of dynamic range with excessive development time in sliver staining
Development time: good spot, saturation, donut
Fluorescent stain
- Dyes are fluorescent on association with SDS-protein complexes (SYPRO Red and SYPRO Orange)
- Requires the use of a laser imaging system
- Able to detect 1-10ng of proteins
Newer fluorescent dyes
Newer fluorescent dyes are metal-chelate dyes that interact with the proteins (SYPRO Rose and SYPRO Ruby)
Detection of fluorescent dye stained proteins
Laser imaging system
How to use fluorescent dye staining for PTM detection in proteins
Some fluorescent dyes are able to pick out specific groups of proteins – phosphoproteins, glycoproteins etc.
(e.g. Pro-Q-diamond (P), Pro-Q-emerald (Gly))
Can combine dyes to track total protein content
distribution/expression of proteins with post-translational modifications.
Multiplexing of fluorescent staining
The Pro-Q Diamond phosphoprotein gel stain,
Pro-Q Emerald glycoprotein gel stains and SYPRO Ruby protein gel stain
—which we have optimized to complement each other in selectivity, sensitivity and staining protocols
—can be used in serial detection of phosphoproteins, glycoproteins and total proteins on a single protein sample separated by 1D or 2D gel electrophoresis
Multiplexing of fluorescent dyes
Pro-Q diamond stain + Pro-Q emerald stain = SYPRO ruby stain
2D-fluorescence difference gel electrophoresis
The dyes are all charge-matched and molecular mass-matched to prevent alterations of pI, and minimize dye-induced shifting of labelled proteins during electrophoresis
process of 2D-DIGE using CY dyes
Protein extracts coming from cells treated under different conditions are marked by different cyanine (Cy) fluorescent dyes
These extracts will then be resolved on the same 2D-PAGE gel
The different level of expression of each protein in the different extracts will then be visible by measuring which color is more present in each spot
Pooled internal standard (label with Cy2)
Protein extract 1 label with Cy3
Protein extract 2 label with Cy5
Mix labelled extracts
2-D electrophoresis
Imaging shows Cy2, Cy3 and Cy5
Image analysis
Principle of 2D-DIGE
Each protein extract and internal standard is labelled with a spectrally distinct fluorescent cyanine (Cy) dye
- Each Cy dyes is charge-matched and have the same molecular mass
- Different Cy dyes are spectrally distinct i.e. have different excitation and emission wavelengths
The labelled protein extracts and internals standard are mixed and run together on the same 2D-PAGE gel
After the run, different fluorescent images of the same gel are obtained and superimposed to detect differences
- Quantitation of fluorescent spots using internal standards allow for determination of differential protein expression in the different protein extracts
Advantage of 2D-DIGE over 2D-PAGE
Increased accuracy in the quantitation of protein spots.
Number of gels required for a specified level of precision is reduced due to low gel-to-gel variation.
Experiments with large sample sizes are feasible.
Comparison of staining methods among Coomassie, fluorescent and sliver
Fluorescent dye and sliver staining show almost similar sensitivity
Coomassie staining has the lowest sensitivity
Comparison of dynamic linearity between staining methods
Range of dynamic linearity : Sliver < fluorescence
Better linearity over a range of values shows more accurate quantification
Panceau S staining
Reversible red stain used to detect proteins on blots
Useful diagnostic tool for western blot to determine if transfer has been successful
Easily removed by washing with water
Zinc imidazole negative staining
Proteins bind to zinc ion via negatively charged amino acids
Imidazole reacts with unbound zinc ion to form a salt complex
The free salt complex (not bound to proteins) containing SDS, imidazole and zinc becomes a precipitate in the gel, forming a dark background
Proteins in the gel interact with salt and do not precipitate easily thus forming lighter regions on the darker background
Zinc imidazole negative staining advantage
Sensitivity is 1 to 10 ng (better than Coomassie staining).
Zinc imidazole negative staining disadvantage
Besides proteins, zinc ions also binds other biopolymers such as nucleic acids and polysaccharides, thus causing high ‘background’ signal.
Epitope tag staining
Dyes that bind to specific amino acid sequences (epitope tag) fused to the ends of recombinant proteins.
Epitope tags = short peptide sequences fused to the end of a recombinant protein to facilitate purification. Imagine a vector containing the gene of interest and epitope tag on the N or C terminus of the gene of interest. Epitope tagged proteins can be recognized by specific antibodies bound to an affinity matrix
Allows monitoring of protein expression in-gel without the need for the lengthy process of Western blotting.
Epitope-tag staining (Oligohistidine tag)
Dyes binds to a specific 6x His tag.
Detects up to 25 ng of a protein fused with a 6xHis tag
Similar concept to use of 6x His tag to purify proteins
using a Ni-NTA (nickel-nitrilotriacetic acid) column.
Nitrilotriacetic acid (NTA) chelates nickel ions which are then bound by oligohistidines domains.
Oligohistidine tag making
N-terminal or C-terminal fusion
PCR product of your gene of interest with in-fusion ends is inserted into the tag
Oligohistidine tag (purification of His-tag proteins)
Purification of His-Tag proteins
Matrix - spacer arm - nitrilotriacetic acid - nickel - protein - Oligo-histidine domain
What is a Oligohistidine tag (staining with labelled NTA)
6X his tag oligohistidine domain bound with nitrilotriacetic acid and fluorophore/enzyme
proteins have a 6x His Tag, this 6x His Tag is bound to nickel and nitrilotriacetic acid labelled with a fluorophore/enzyme
Immunoblotting process
antigen samples are loaded into a separation gel,
blotting tank is used to transfer the proteins to the nitrocellulose membrane
the nitrocellulose membrane is immunoblotted with labelled antibodies
autoradiography is carried out, develop and fix autoradiograph
antigen bands are visualized
Western blot / immunoblot principles
Electrophoretic or capillary transfer of protein molecules onto the surface of an immobilizing membrane
The absorbed proteins are free to bind with macromolecules like antigens, antibodies, lectins and DNA
Applicable to IEF, SDS-PAGE and 2D-PAGE gels
Capillary blotting
Diffusion carries the proteins across to the membrane
electroblotting transfer stack
current flows from the bottom negatively charged stack to the top positively charged stack
in sequence from the bottom, negative layer, sponge,, filter paper, membrane gel, filter paper, sponge and the top positive layer
Western blotting process
in between the SDS-PAGE gel and the blotting paper is a membrane. There is a blotting paper on top of the gel to soak up excess. the current flows down and drives the protein to migrate on to the membrane
the membrane add 1 antibody, wash excess , then the 2nd antibody, wash excess, then add substrate then visualize the color change
2 choices of membranes to use
Nitrocellulose
PVDF (polyvinylidenedifluoride)
Nitrocellulose principles
Has a high affinity for proteins.
Hydrophobic and electrostatic interactions with amino acids.
Wetting of membrane with water.
PVDF (polyvinylidenedifluoride) principles
Proteins bind by hydrophobic interactions.
Loading protein capacity per unit area higher than nitrocellulose.
Higher mechanical strength than nitrocellulose.
Wetting of membrane with methanol.
Better choice than nitrocellulose if reprobing is needed
3 choices of antibodies
Polyclonal antibody, Monoclonal antibody, Cross reactivity antibody
they are antibodies produced by B cells as part of immune response against foreign antigen
Polyclonal antibodies
mixture of different antibodies that bind to different epitopes of the antigen.
Monoclonal antibodies
single type of antibody that recognises one specific epitope of the antigen
Note about choice of antibodies
Cross reactivity with other proteins and across species can reduce accuracy of experimental results.
With other proteins and across species can reduce accuracy of experimental results.
Blocking introduction
Use of macromolecules that do not take part in the visualisation step to block free binding sites on the membrane.
Usually a solution of 2-10% bovine serum albumin (BSA) or 5% non-fat milk in PBST (phosphate buffered saline + 0.05% Tween-20).
2 reasons why blocking is needed
It prevents antibodies from attaching non-specifically to the membrane.
It reduces background, thus increasing signal to noise ratio.
blocking reagent sticks to the membrane almost completely and blocks antibodies from attaching
What if there is no blocking
Increased background signal and non-specific binding will occur
Colorimetric detection
depends on incubation of the immunoblot with a substrate dye that reacts with the reporter enzyme bound to a secondary antibody.
- This converts the soluble dye into an insoluble precipitate of different color.
- This detection does not allow for reprobing of the immunoblot as the blot is permanently stained.
Chemiluminescence detection
depends on incubation of the immunoblot with a substrate which reacts with the reporter enzyme bound to a secondary antibody.
- The substrate emits luminescence when it is catalysed.
- This detection allows for reprobing as the substrate does not bind to the
Comparison between colorimetric and chemiluminescence
Colorimetric
Permanent colour on membrane
- Alkaline phosphatase/
- Horse radish peroxidase
- No reprobing possible
Chemiluminescence
- Emitted light is shortlived.
- Alkaline phosphatase/
- Horse radish peroxidase
- Reprobing possible
Detection of glycosylation with western blotting introduction
With glycosylation, the size of the protein is significantly increased.
Due to heterogeneity of glycosylation, a smear of a protein band is observed if glycosylation of the protein has occurred
Detection of phosphorylation with western blotting
Multiple bands differing by <10kDa
Intensity of bands may vary due to different proportion of phosphorylated proteins.
Phosphorylation adds weight to the protein, which can be detected on the gel if the same protein had multiple bands differing by <10kDA. However, the intensity of bands may vary due to different proportion of phosphorylated proteins.
Detection of disulfide bonding with Western blotting
Disulphide bonding can be detected on non-reducing SDS-PAGE.
On addition of DTT in the SDS-PAGE sample buffer, the disulphide bonds are broken, resulting in a single band.
Run the proteins on 2 gels, one gel with DTT and another gel without DTT, if oligomers or aggregates are present on the gel without DTT, disulphide bonding of the proteins has occurred.
Comparative analysis of 2D-PAGE gel
Differentiate spots based on their intensity
Darker spots can imply overexpression of proteins, higher amount of expressed proteins, potentially a product of oncogene
5 steps sequence of comparative analysis in 2D-PAGE
Scanning of image - convert gel spots to digital data, use densitometry to measure intensity
Image Processing - gaussian curves, data smoothing, enhance contrast and substract background
Spot detection - automated or manual, sensitivity and normalization
Gel matching - compare identical spots and use landmarks to compare
Data analysis - quantification, 3D image of spots and interpretation
How the densitometer scans the image
Convert ‘analog’ spots on gel into digital data
Analog means ‘something that is similar or comparable to something else’
Densitometers obtains high resolution images
Used for wet or dried gels that have been stained, X-ray films and blots.
It measures intensity of all areas of the gel image.
Densitometry
It measure intensity of the protein bands. Peaks represent the bands
Raw intensity readings of the peaks will include
- Unequal intensity of background
- Baseline is not level (Baseline is the line below peaks)
What is background in 2D-PAGE
Background is intensity of the area surrounding spots or bands
Image processing
Digital data converted into Gaussian curves.
Algorithms are used to smoothen curve, removing statistical noise (via mathematical manipulation).
Contract enhancement
To see distinct spots
Contrast enhancement performs simple pattern recognition to identify features that resemble dark circular spots. It enables identification and detection of spots.
Background subtraction
To remove meaningless changes in the background intensity of the gel.
Hence, it enables accurate quantitation of the intensity of the spots
Contrast enhancement
Contrast enhancement performs simple pattern recognition to identify features that resemble dark circular spots.
It enables easier identification and detection of spots.
How the use of background subtraction changes the baseline ?
Sloping baseline before processing
Level baseline after processing
Automated Spot detection
Automatic detection aided by manual input
- User defines the spots.
Need to adjust sensitivity on automated spot detector.
Why need to adjust sensitivity on automated spot detector
Too little sensitivity = missed spots
Too much sensitivity = false positives
Gel matching
Gel matching involves the comparison of identical spots on different gels
Disadvantages of gel matching
Matching is seldom 100% due to variations in experimental techniques (staining, gel preparation).
What is the use of landmarks
Use of landmarks to improve matching
Landmarks are spots that are large or have high intensity
Spot normalization
Normalization is the process of reducing unwanted variation between spots on a gel.
Qualitative data analysis
Summary of comparative analysis in 2D-PAGE
Analog to digital data Scanning of image Densitometry - measure intensity
Gaussian curves data smoothing Image Processing Enhance contrast Substract background
Automation Manual Spot detection Sensitivity Normalization
Compare identical spot Gel matching Landmarks
Quantification, 3D-image of spots Data analysis Interpretation
Summary
- 2D-PAGE
Staining methods used in 2D-PAGE
Coomassie, Silver, Fluorescent in 2D-DIGE
Ponceau S, Zinc imidazole, Epitope-tag - Western blotting
- Comparative analysis
