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Genomic and Proteomics > Chapter 7 2D PAGE > Flashcards

Chapter 7 2D PAGE Flashcards

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1
Q

Objectives

A

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

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2
Q

Things to do in workflow in proteomic analysis

A

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

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3
Q

6 staining methods for 2-PAGE analysis

A

Coomassie blue

Silver staining

Fluorescent

Ponceau S

Zinc Imidazole

Epitope tag

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4
Q

Coomassie blue dye introduction

A

Based on the binding of the Coomassie brillant blue dye (G-250 and R-250) to proteins

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5
Q

Principle of Coomassie blue dye staining

A

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).

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6
Q

Conventional Commasie blue dye

A

Able to detect 30-100ng of proteins

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7
Q

Comparison of commasie blue staining compared to other

A

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

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8
Q

How to increase Coomassie blue staining sensitivity

A

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
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9
Q

Comparing CCB G-250 and CCB R-250

A

CCB G-250 is blue

CCB R-250 is pink

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10
Q

Conventional CCB effect

A

Staining time: 1 hour, no bands visible before de- staining

After de-staining: 1 hour in methanol, acetic acid

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11
Q

Colloidal CCB effect

A

Staining time: 1 hour bands visible in the staining tray

After de-staining, 1 hour water wash enhancement

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12
Q

Sliver stain introduction

A

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

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13
Q

3 limitations of sliver stain

A
  1. Not an end-point method
    The amount of development time needs to be fixed
    Too short => loss in sensitivity
    Too long => over-staining
  2. Relationship between silver and protein
    - Narrow linear dynamic range
  3. Not compatible with analysis by Mass Spectrometry
    - Formaldehyde can cross-link proteins to gel matrix
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14
Q

Sliver stain

A

Loss of dynamic range with excessive development time in sliver staining

Development time: good spot, saturation, donut

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15
Q

Fluorescent stain

A
  1. Dyes are fluorescent on association with SDS-protein complexes (SYPRO Red and SYPRO Orange)
  2. Requires the use of a laser imaging system
  3. Able to detect 1-10ng of proteins
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16
Q

Newer fluorescent dyes

A

Newer fluorescent dyes are metal-chelate dyes that interact with the proteins (SYPRO Rose and SYPRO Ruby)

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17
Q

Detection of fluorescent dye stained proteins

A

Laser imaging system

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18
Q

How to use fluorescent dye staining for PTM detection in proteins

A

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.

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19
Q

Multiplexing of fluorescent staining

A

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

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20
Q

Multiplexing of fluorescent dyes

A

Pro-Q diamond stain + Pro-Q emerald stain = SYPRO ruby stain

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21
Q

2D-fluorescence difference gel electrophoresis

A

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

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22
Q

process of 2D-DIGE using CY dyes

A

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

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23
Q

Principle of 2D-DIGE

A

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

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24
Q

Advantage of 2D-DIGE over 2D-PAGE

A

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.

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25
Comparison of staining methods among Coomassie, fluorescent and sliver
Fluorescent dye and sliver staining show almost similar sensitivity Coomassie staining has the lowest sensitivity
26
Comparison of dynamic linearity between staining methods
Range of dynamic linearity : Sliver < fluorescence Better linearity over a range of values shows more accurate quantification
27
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
28
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
29
Zinc imidazole negative staining advantage
Sensitivity is 1 to 10 ng (better than Coomassie staining).
30
Zinc imidazole negative staining disadvantage
Besides proteins, zinc ions also binds other biopolymers such as nucleic acids and polysaccharides, thus causing high ‘background’ signal.
31
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.
32
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.
33
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
34
Oligohistidine tag (purification of His-tag proteins)
Purification of His-Tag proteins | Matrix - spacer arm - nitrilotriacetic acid - nickel - protein - Oligo-histidine domain
35
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
36
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
37
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
38
Capillary blotting
Diffusion carries the proteins across to the membrane
39
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
40
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
41
2 choices of membranes to use
Nitrocellulose PVDF (polyvinylidenedifluoride)
42
Nitrocellulose principles
Has a high affinity for proteins. Hydrophobic and electrostatic interactions with amino acids. Wetting of membrane with water.
43
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
44
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
45
Polyclonal antibodies
mixture of different antibodies that bind to different epitopes of the antigen.
46
Monoclonal antibodies
single type of antibody that recognises one specific epitope of the antigen
47
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.
48
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).
49
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
50
What if there is no blocking
Increased background signal and non-specific binding will occur
51
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.
52
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
53
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
54
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
55
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.
56
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.
57
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
58
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
59
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.
60
Densitometry
It measure intensity of the protein bands. Peaks represent the bands
61
Raw intensity readings of the peaks will include
- Unequal intensity of background | - Baseline is not level (Baseline is the line below peaks)
62
What is background in 2D-PAGE
Background is intensity of the area surrounding spots or bands
63
Image processing
Digital data converted into Gaussian curves. Algorithms are used to smoothen curve, removing statistical noise (via mathematical manipulation).
64
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.
65
Background subtraction
To remove meaningless changes in the background intensity of the gel. Hence, it enables accurate quantitation of the intensity of the spots
66
Contrast enhancement
Contrast enhancement performs simple pattern recognition to identify features that resemble dark circular spots. It enables easier identification and detection of spots.
67
How the use of background subtraction changes the baseline ?
Sloping baseline before processing Level baseline after processing
68
Automated Spot detection
Automatic detection aided by manual input - User defines the spots. Need to adjust sensitivity on automated spot detector.
69
Why need to adjust sensitivity on automated spot detector
Too little sensitivity = missed spots Too much sensitivity = false positives
70
Gel matching
Gel matching involves the comparison of identical spots on different gels
71
Disadvantages of gel matching
Matching is seldom 100% due to variations in experimental techniques (staining, gel preparation).
72
What is the use of landmarks
Use of landmarks to improve matching Landmarks are spots that are large or have high intensity
73
Spot normalization
Normalization is the process of reducing unwanted variation between spots on a gel. Qualitative data analysis
74
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
75
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

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