Detection techniques for gel-based proteomics

Detection techniques for gel-based proteomics

Harini ChandraAffiliations

Detection techniques using organic and fluorescent dyes for gel-based proteomics have developed

significantly over the last few years allowing researchers to carry out high-throughput studies with

increased sensitivity.

Master Layout

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1Coomassie Brilliant Blue (CBB)

Silver staining

SYPRO Ruby Red

SYPRO orangeCyanine dyes

Pro-Q-diamond

Lightining fast

Gel-based detection techniques

Definitions of the components:

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1 1. Coomassie Brilliant Blue (CBB): Coomassie blue dyes (R-250 & G-250) are low cost, organic dyes that are easy to use for staining of proteins that have been separated by electrophoretic techniques. Gels are soaked in the dye solution dissolved in methanol and acetic acid, after which excess stain is then washed off with a destaining solution. The higher affinity of proteins towards the dye molecules, allows the protein bands to be selectively stained with sensitivity of 8-100 ng without significant staining of the background. These dyes are also compatible for further MS-based applications.

2. Silver staining: Electrophoresis gels are saturated with silver ions in the form of either silver nitrate or as an ammonia-silver complex after fixing the proteins in the gel. The less tightly bound metal ions are subsequently washed off and the protein-bound silver ions are reduced to metallic silver using formaldehyde under alkaline conditions in presence of sodium carbonate or citrate buffer solution. Although as little as 1 ng of protein can be detected by silver staining, the gel-to-gel reproducibility remains an issue. Compatibility of silver stains with MS is another issue, which has however, been overcome in recently introduced silver stains.

3.SYPRO Ruby Red: This is a ruthenium-based metal chelate fluorescent stain that provides a single step protein staining procedure with low background staining in polyacrylamide gels. They have been observed to be as sensitive as the silver stains (0.25-1 ng) with the liner dynamic range extending over three orders of magnitude, thereby showing better performance than CBB and silver stains. This stain can also be combined with other dyes thereby allowing multiple detections in a single gel.

4. SYPRO orange: This dye is less sensitive than SYPRO Ruby Red but is also capable of detecting proteins in SDS-PAGE gels in a rapid single step process without the requirement for any destaining procedure. As little as 4-30 ng of protein can be detected by this fluorescent dye and it is compatible for further MS-based applications. Two other similar dyes having comparable sensitivities and similar excitation and emission wavelengths are SYPRO Red and Tangerine.

Definitions of the components:

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15. Cyanine dyes: They are water-soluble derivatives of N-hydroxy succinimide that covalently bind the -amino groups of a protein’s lysine residues and are spectrally resolvable as they fluoresce at distinct wavelengths. The labelled protein samples can therefore be mixed and run on a single gel, thereby eliminating the problem of gel-to-gel variations, the principle employed in difference gel electrophoresis (DIGE). Cy3, Cy5 and Cy2 having sensitivities of 0.1-2 ng are most commonly used for proteomic and MS-based applications.

6. Lightning fast/Deep purple: A fluorescence-based stain obtained from the fungus Epicoccum nigrum that can be used for detecting proteins in 1-D and 2-D gel electrophoresis with sensitivity down to 100 pg protein. Stained proteins are excited by near-UV or visible light with maximum fluorescence emission occurring at around 610nm. These dyes are suited for further use with Edman or MS applications.

7. Pro-Q-Diamond: This fluorescent dye is capable of detecting modified proteins that have been phosphorylated at their serine, threonine or tyrosine residues. They are suitable for use with electrophoretic techniques or with protein microarrays and offer sensitivity down to few ng levels, depending upon the format in which they are used. This dye can also be combined with other staining procedures thereby allowing more than one detection protocol on a single gel.

Step 1:

Action Audio Narration

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4Description of the action

Coomassie Brilliant Blue (CBB)

Silver staining

SYPRO Ruby Red

SYPRO orangeCyanine dyes

Pro-Q-diamond

Lightining fast (Deep purple total protein stain)

Each technique shown must appear one at a time as depicted.

First the central heading must be shown followed by appearance of each of the arrows and the boxes. User must be allowed to click on these headings to read more about them as given in the previous two slides. Finally the titles in the blue, grey, green, yellow and red boxes must be highlighted.

As described in previous two slides for each of the headings.

Gel-based detection techniques

Step 2:


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Completed electrophoresis gel

Coomassie blue solution

Tray Mechanical rocker

Show the tray on the left followed by a blue solution in it and placement of the ‘gel’ in the solution.

(Please redraw chemical structure and mechanical rocker)First show the tray on the left followed by a blue solution being filled in it. Next show the ‘completed gel’ being placed in this solution. Then show the instrument on the right and the tray with gel being placed on it. This must then be shown to rock from side to side as indicated by the arrow.

The completed electrophoresis gel is placed in a tray containing the Coomassie blue staining solution, typically R-250, that has been dissolved in an aqueous solution of ethanol and acetic acid. Negatively charged Coomassie dye interacts with proteins through ionic and non-covalent interactions. This tray is then placed on a mechanical rocker that allows for uniform contact of the gel with the solution by means of gentle rocking.

Forms ionic & non-covalent interactions with proteins

http://en.wikipedia.org/wiki/File:Coomassie_Brilliant_Blue_G-250.svg

Step 3:


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Stained gel

Destaining solutionTray

A 2-D gel image showing final gel pattern with stained protein bands

Excess stain removed

Show the ‘gel’ being removed from the blue solution and being placed in a transparent solution as shown above.

First show the gel being removed from the blue solution in the previous slide and being placed in a colorless solution, as shown above. This must again be placed on the ‘mechanical rocker’ shown above and as the rocking takes place, the colorless solution must slowly turn blue.

The stained gel is then placed in a destaining solution consisting of 50% methanol & 10% acetic acid to remove any excess dye that may be bound to the gel. The gel is finally removed and viewed by means of a scanner where the blue protein bands can be easily detected.

Step 4:


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Fixing solution (acid/alcohol) – to minimize diffusion of protein bands in gel


Show the tray on the left followed by first a pale grey solution and then a pale orange solution in it.

First show the tray on the left followed by a pale grey solution being filled in it and the label ‘fixing solution’. Next show the ‘completed gel’ being placed in this solution. After a while, show the grey solution being removed and being filled by a pale orange solution, ‘silver stain’. When this is done, the grey circles must appear in solution. Then show the instrument on the right and the tray with gel being placed on it. This must then be shown to rock from side to side as indicated by the arrow.

The completed gel is first placed in a fixing solution of methanol and acetic acid that fixes the protein bands in the gel and minimizes any diffusion. This must be subjected to gentle shaking for around 30 minutes after which the silver stain solution is added. The gels are rocked gently in order to allow for proper and uniform staining.

Silver staining

Silver stain

Ag+

Ag+

Ag+

Ag+

Ag+

Step 5:


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Stained gel

Developing solution

Final gel pattern with silver stained protein bands

Show the ‘gel’ being removed from the orange solution and being placed in the pale pink solution.

First show the gel being removed from the orange solution in the previous slide and then being placed in a pale pink solution containing green ovals and orange rectangles along with the grey circles, as shown above. This must again be placed on the ‘mechanical rocker’ shown in the previous slide and rocked gently. When this is taking place, the equation in the grey box must appear. Finally the image on the right must be shown.

The silver stained gels are first washed to remove any excess stain and then placed in a developing solution where the silver ions get reduced to metallic silver. Formaldehyde in an alkaline solution in presence of sodium carbonate or other alkaline buffers are commonly used for this process. This gel can then be viewed as dark bands against a light background as shown in the figure on the right.

Silver staining

Ag+

Ag+

Ag+

Ag+

Ag+

Ag+

HCHO

HCHO

HCHO

OH-

OH-

OH-

Ag+ + HCHO + OH- Ag+ + HCOO- + H2O

Step 6:


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Fixing solution 50% methanol+7% acetic acid


As shown in animation.

First show the tray on the left with a pale grey solution in it and the label ‘fixing solution’. Next show the ‘completed gel’ being placed in this solution. After a while, show the grey solution being removed and being filled by a pale red solution, ‘SYPRO Ruby red’. Then show the instrument on the right and the tray with gel being placed on it. This must then be shown to rock from side to side as indicated by the arrow.

The completed gel is first placed in a fixing solution of methanol and acetic acid that fixes the protein bands in the gel and minimizes any diffusion. This must be subjected to gentle shaking for around 30 minutes after which the gel is soaked in fluorescent SYPRO Ruby Red stain solution. The gel is rocked gently for uniform staining with the ruthenium-based chelate dye.

SYPRO Ruby Red stain solution

SYPRO Ruby Red

Step 7:


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Show the ‘gel’ being removed from the red solution and being placed in the pale blue solution.

(Please redraw all figures.)First show the gel being removed from the red solution in the previous slide and then being placed in a pale blue solution as shown above. This must again be placed on the ‘mechanical rocker’ shown in the previous slide and rocked gently. The gel must be removed and placed below the instrument shown on top right but the pattern must not be visible. Then the violet arrows indicating light must be shown to enter the instrument as indicated followed by the red beam coming out and the gel being displayed with its pattern.

The gel can be left overnight in the dye solution and then washed with a methanol-acetic acid solution. The MS-compatible gel is then viewed using UV or Laser-scanning instruments with an excitation wavelength near 280 or 450 nm. The stain has two excitation maxima and an emission maxima of 610 nm.

SYPRO Ruby Red

Decreasing molecular weight

Final SYPRO Ruby Red stained gel pattern

Decreasing pH

Washing solution – 10% methanol+7% acetic acid

Gel scanner

Maximum excitation wavelength – 280 nm / 450 nm

Emission maxima – 610 nm

Step 8:


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In this detection technique, the dyes are mixed with the samples prior to electrophoresis. Each protein sample as well as the internal standard is labeled with a differently fluorescing cyanine dye which allows all protein samples to be simultaneously run on a single 2-DE gel.

Cyanine dyes

Cy3

Cy5

Cy2

(Please redraw all figures.)First show the ‘sample 1’ tube followed by the red dye which must mix with it to give the red tube. Then the ‘sample 2’ tube mixing with the blue dye and then ‘sample 3’ mixing with the green dye. These 3 colored tubes must then be mixed to give the violet tube. The micropipette must be dipped in the purple solution and the yellow tip must become purple. It must then move to the blue strip and a small purple layer must appear on top of the circle on the blue strip. There must be formation of bands in the strip which must move in the direction indicated and the figure on right must be shown.

Sample 1

Sample 2

Internal standard (Sample 1+2)

IEF in progress IPG strip

Step 9:


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As shown in the animation.

Cy5 excitation wavelength

Only Cy3 labeled proteins

Only Cy5 labeled proteins



DIGE Imager

All proteins with Cy2 label

SDS-polyacrylamide gel slab

Cyanine dyes

(Please redraw all figures.)First show the horizontal blue strip and the blue slab below. The strip must move down and fit into the slot below. The blue bands must be shown to be moving in the direction indicated. The instrument on top right must then be shown followed by the appearance of the three beams with their respective blue figures and labels below.

This gel can be viewed by illuminating it alternately with excitation wavelengths corresponding to the various Cy dyes. This technique minimizes gel-to-gel variations and allows a large number of samples to be run using fewer gels, thereby proving to be extremely useful for large-scale proteomic applications.

IPG strip

Step 10:


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ProQ-diamond is a useful technique for detection of phosphoproteins and when used in combination with SYPRO-Ruby red, it gives a good comparative profile of the total protein content and the phosphoprotein content. The procedure for staining is similar to SYPRO-Ruby Red as discussed earlier. This fluorescent dye offers sensitivity down to ng levels and detects phosphorylations taking place at serine, threonine and tyrosine residues.

First show the first two trays on the left with the solution in it. The gels must be placed in these two trays. Next, show this solution being removed and the next solution as shown in middle panel being added to the tray. Finally, this solution must also be removed and the last blue solution must be added to the tray.

Pro-Q-diamond

1. Fixing (methanol+acetic acid)

ProQ diamond stain

2. Staining 3. Washing (methanol+acetic acid)

SYPRO-Ruby Red

Dual staining: Pro-Q-Diamond and SYPRO Ruby Red

Step 11:


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As shown in the animation.

(Please redraw all figures.)First show the instrument on top followed by the yellow beam and the gel placed below that. This must be followed by the graph on the top right. Next, show the red beam coming out of the instrument with its gel below and the second graph on bottom right.

The two stained gels can be scanned at their respective excitation wavelengths for detection of total protein and phosphoprotein images. The gel stained with Pro-Q-diamond will show fewer bands than the SYPRO-Ruby stained gel due to fewer number of phosphoproteins in the total protein mixture. Hence, a good comparative analysis can be done using this protocol and the stained gel can be used for further MS-based applications

Decreasing molecular weight

Decreasing pH

Gel scanner


Phosphoprotein image

Total protein image by SYPRO-Ruby Red


Phosphoprotein image

Total protein image

Pro-Q-diamond: Dual staining

Interactivity option 1:Step No:1

Boundary/limitsInteractivity Type Results

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Coomassie Blue

Silver stain

SYPRO-Ruby Red

SYPRO-Orange

Cyanine dyes

Lightning fast

Pro-Q-diamond

Sensitivity (ng)

8-100 1 0.25-1 4-30 0.1-2 <1 1-5

Compatibility with MS

Mostlyincompatible

Drag and drop.

The rows must be displayed one at a time to the user along with its input values. User must drag and drop the inputs in the table into the correct column.

OptionsOnce the user drags all the inputs into their correct cells as shown above, the message ‘Well done!’ must be displayed and user is re-directed to step 2.

Interactivity option 1:Step No:2 1

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Coomassie Blue

Silver stain

SYPRO-Ruby Red

SYPRO-Orange

Cyanine dyes

Lightning fast (Deep purple)

Pro-Q-diamond

Merits Easy to use, good reproducibility

Good sensitivity

Good sensitivity, reproducibility, single step process, wider linear dynamic range

Convenient to use, good sensitivity

Allow multiplexing, very sensitive

Natural product, highly sensitive, good linear dynamic range

Good sensitivity for phosphoprotein detection, very selective

Demerits

Poor sensitivity, small linear dynamic range

Poor reproducibility, narrow linear dynamic range

Excessive gel swelling possible if cross-linker concentration is low.

Problems in electroblotting, enzyme activity measurement due to 7% acetic acid

Expensive Relatively less sensitive in detecting total number of protein spots.

Suitable only for PTM staining

Questionnaire1

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1. Which of the following detection methods is mostly not compatible for MS applications?

Answers: a) SYPRO-Ruby red b) Pro-Q-Diamond c) Silver staining d) SYPRO-Tangerine

2. The emission maxima of Pro-Q diamond is

Answers: a) 610 nm b) 580 nm c) 480 nm d) 250 nm

3. Which of the following detection stains is obtained from a natural source?

Answers: a) Coomassie blue b) SYPRO-Orange c) Cyanine dyes d) Deep purple/Lightning

fast

4. If a Pro-Q-labeled gel shows 8 bands and a SYPRO-Ruby red-labeled gel shows 36 bands,

then how many non-phosphorylated proteins are present in the mixture?

Answers: a) 44 b) 8 c) 26 d) 28

Links for further reading

Reference websites:Steinberg, T. H. et al. Pro-Q Diamond phosphoprotein stain: a new reagent for detection of

phosphoproteins and phosphopeptides in polyacrylamide gels and in microarrays.

Research papers: Patton, W. F. Detection technologies in proteome analysis. J. Chromatogr. B. 2002, 771, 3-31.

Mackintosh, J. A. et al. A fluorescent natural product for ultra sensitive detection of proteins in one-

dimensional and two-dimensional gel electrophoresis. Proteomics 2003, 3, 2273-2288.

Johnson, C. J., Zhukovsky, N., Cass, A. E. G., Nagy, J. M. Proteomics, nanotechnology and molecular

diagnostics. Proteomics 2008, 8, 715-730.

Berggren, K. N. et al. An improved formulation of SYPRO Ruby protein gel stain: Comparison with the

original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation. Proteomics

2002, 2, 486-498.

Chakravarti, B., Ratanaprayul, W., Dalal, N., Chakravarti, D. N. Comparison of SYPRO Ruby and Deep

Purple Using Commonly Available UV Transilluminator: Wide-Scale Application in Proteomic Research. J

Proteome Res. 2008, 7(7), 2797-802.

Detection techniques for gel-based proteomics

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