Protein Staining in Polyacrylamide Gels with Rapid and Sensitive Colloidal Coomassie G-250

Summary

This video script outlines a modified Kumasi staining protocol for detecting proteins in polyacrylamide gels, using two-dimensional gel electrophoresis as an example. It details the process of isoelectric focusing and SDS-PAGE, emphasizing the protocol's high sensitivity, cost-effectiveness, and practicality for analytical proteomics. The script guides through the preparation of reagents, staining, and destaining, highlighting the importance of using high-quality chemicals and thorough washing for optimal results.

Takeaways

• 🧪 Modified Kumasi staining protocol is demonstrated for detecting proteins in polyacrylamide gels.
• 🎯 The example of two-dimensional gel electrophoresis is used to illustrate the procedure.
• 🌟 The first dimension involves isoelectric focusing (IEF) using immobilized pH gradient (IPG) strips.
• 🥼 Protein samples are applied via anodic cup loading during the focusing process.
• 🧫 The second dimension is performed by SDS-PAGE for separation based on molecular weight.
• 💧 After electrophoresis, proteins are detected by staining with colloidal Kumasi.
• 👩‍🔬 Nadine Dubala from the University of Dusseldorf emphasizes the protocol's advantages in sensitivity, cost, and practicality.
• 🔬 The method is particularly useful in analytical proteomics for myocardial protein expression analysis.
• 🛠️ Proper preparation of the staining solution is crucial, with sequential addition of components.
• 🧼 Efficient washing of gels post-SDS-PAGE is necessary to remove residual SDS for effective staining.
• 📈 The protocol can detect as low as one nanogram of protein, making it a valuable tool for proteomics research.

Q & A

What is the primary focus of the video?

The primary focus of the video is to demonstrate the power of a modified Kumasi staining protocol for detecting proteins in polyacrylamide gels, specifically using the example of two-dimensional gel electrophoresis.

What is the first dimension separation in 2D gel electrophoresis?

The first dimension separation in 2D gel electrophoresis is isoelectric focusing (IEF), where immobilized pH gradient (IPG) strips are used to separate proteins according to their isoelectric points.

How are the IPG strips prepared for protein sample application?

The IPG strips are prepared by rehydrating them individually in 125 microliters of rehydration solution using the Immobiline dry strip re-swilling tray for at least 10 hours.

How are the protein samples prepared for the IEF?

Protein samples are prepared by dissolving TCA-precipitated protein samples from different heart preparations in IEF sample buffer to a concentration of 100 micrograms protein per 100 microliters, with solubilization taking at least 30 minutes at room temperature on a vortexer.

What is the role of the cooling unit during IEF?

The cooling unit is set to 20 degrees Celsius to help maintain a consistent temperature throughout the IEF process, which is crucial for the proper separation of proteins.

How are the proteins separated in the second dimension of 2D gel electrophoresis?

The second dimension separation is performed by SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) on a vertical electrophoresis system, where proteins are separated for 2.5 hours starting at 80 Volts for 15 minutes, followed by 120 Volts until the dye front reaches the bottom of the gel cassette.

What are the critical steps in preparing the colloidal Kumasi staining solution?

The critical steps include dissolving aluminum sulfate, intermixing ethanol and adding CBB G250, followed by the addition of orthophosphoric acid to aggregate the Kumasi molecules into their colloidal state, and finally adjusting the volume with Millie Q water without filtering.

Why is it important to wash the gel thoroughly before staining?

Thorough washing of the gel is important to remove residual SDS, which can disrupt the binding of the dye to the protein and cause poor sensitivity in the staining process.

What is the recommended incubation time for the Kumasi staining to achieve close to 100% staining?

While 80 to 100% of the staining can be accomplished within two hours of incubation, an overnight incubation is recommended to achieve close to 100% staining.

How does the sensitivity of the Kumasi staining compare to other staining methods?

The Kumasi staining is comparable to silver staining, which is reported to be of high sensitivity and compatibility with mass spectrometry, making it a good alternative for non-labeling methods in gel-based proteomics.

What are the key factors to consider when using the Kumasi staining procedure?

Key factors include using analytical grade chemicals and water for purity, the sequential addition of components to the staining solution, and efficient washing of residual SDS from the gels after electrophoretic separation to ensure optimal results.

Outlines

00:00

🧬 Introduction to Modified Kumasi Staining Protocol

This paragraph introduces the modified Kumasi staining protocol, a method used to detect several nanograms of protein in polyacrylamide gels, with a focus on two-dimensional gel electrophoresis. The process involves rehydration of immobilized pH gradient strips in denaturing buffer, application of protein samples, and separation based on isoelectric points. Nadine Dubala from the University of Dusseldorf emphasizes the protocol's superior features in sensitivity, cost, and practicality for proteomics, especially in detecting myocardial protein expression changes. The first dimension of 2D gel electrophoresis is isoelectric focusing (IEF), where IPG strips are rehydrated and protein samples are prepared for application. The paragraph concludes with the setup for IEF, including the preparation of the immobiline dry strip kit and the application of protein samples into sample cups.

05:01

🧪 Equilibration and Second Dimension Separation

This paragraph discusses the equilibration process involving reduction and alkylation, which prepares the IPG strips for the second dimension of 2D gel electrophoresis. The reduction is performed using diethyothritol equilibration solution, followed by alkylation with Iota acetamide. The strips are then rinsed, blotted, and transferred to 1X SDS running buffer. The second dimension is carried out by SDS-PAGE on vertical electrophoresis systems with 12 percent acrylamide gels. The proteins are separated with a specific voltage regimen. While the proteins are being separated, the paragraph describes the preparation of colloidal Kumasi staining solution, emphasizing the importance of sequential addition of components and the final appearance of colloidal particles in the solution.

10:03

🌟 Staining and Destaining Process

This paragraph details the staining process of the 2D gel with the prepared Kumasi solution. The gel is washed with Millie Q water, followed by staining with the Kumasi solution on a shaker for an extended period. The first protein spots become visible after 10 minutes, with 80-100% staining achieved within two hours. However, an overnight incubation is recommended for optimal results. After staining, the gel is rinsed, and residual dye particles are removed. The destaining process is then described, which enhances the color intensity of the Kumasi stain. The final result should be a distinctly resolved 2D gel with dark blue protein spots, comparable to silver staining. The paragraph concludes by highlighting the protocol's efficiency, low labor requirement, and the ability to detect as low as one nanogram of protein, making it an excellent alternative to non-labeling methods in proteomics.

Keywords

💡Kumasi staining

Kumasi staining is a technique used for visualizing proteins after electrophoretic separations. In the context of the video, it is highlighted for its superior features in sensitivity, cost-effectiveness, and practicality, especially for analytical proteomics. The process involves the use of colloidal particles for staining, which allows for the detection of proteins in polyacrylamide gels with high sensitivity, even as low as several nanograms.

💡Two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis (2D GE) is a method used to separate proteins based on two different properties: isoelectric point (pI) and molecular weight. The first dimension involves isoelectric focusing (IEF) where proteins are separated according to their pI, and the second dimension involves SDS-PAGE where proteins are separated by their molecular weight. This technique is crucial for comparative proteomics and is the main focus of the video demonstration.

💡Isoelectric focusing (IEF)

Isoelectric focusing is the first dimension of 2D gel electrophoresis, where proteins are separated based on their isoelectric points (pI). This process involves the use of immobilized pH gradient (IPG) strips that are rehydrated and then loaded with protein samples. The application of an electric field causes the proteins to migrate to their respective isoelectric points where they carry no net charge and thus focus into distinct bands.

💡SDS-PAGE

SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is the second dimension separation technique used in 2D gel electrophoresis. It separates proteins based on their molecular weight. The proteins from the IEF step are subjected to SDS-PAGE, where they are further separated and resolved on a polyacrylamide gel, allowing for the visualization of distinct protein bands.

💡Immobilized pH gradient (IPG) strips

IPG strips are used in the first dimension of 2D gel electrophoresis for isoelectric focusing. These strips have a pH gradient along their length, which allows for the separation of proteins based on their isoelectric points. The strips are rehydrated with a protein sample, and then subjected to an electric field to focus the proteins.

💡Protein detection

Protein detection refers to the process of visualizing and identifying proteins after they have been separated by techniques such as 2D gel electrophoresis. In the video, colloidal Kumasi staining is used for protein detection, which involves the binding of the dye to the proteins, allowing them to be seen as distinct spots on the gel.

💡Colloidal

Colloidal refers to a state of matter where particles are dispersed evenly throughout a medium, forming a stable suspension. In the context of Kumasi staining, the colloidal state is essential as it allows the staining particles to effectively bind to the proteins within the gel matrix, facilitating the detection and visualization of proteins.

💡Staining

Staining is a process used to color or mark substances for easier visualization or identification. In the context of the video, staining refers to the application of the Kumasi dye to the separated proteins on the gel. This process allows the proteins to be seen as distinct blue spots, making it possible to analyze and compare protein expression patterns.

💡Analytical proteomics

Analytical proteomics is the study of proteins, their structures, functions, and interactions at a large scale. In the video, the Kumasi staining protocol is highlighted for its utility in this field, particularly for detecting changes in myocardial protein expression. The method allows for the sensitive and cost-effective analysis of proteins, which is crucial for advancing research in proteomics.

💡Myocardial protein expression

Myocardial protein expression refers to the levels and types of proteins present in the myocardium, the muscular part of the heart. The video demonstrates how the Kumasi staining protocol can be used to detect and analyze changes in myocardial protein expression, which is important for understanding heart function and disease.

💡Destaining

Destaining is the process of removing excess stain from a gel after protein bands have been visualized. This step is crucial for improving the clarity of the gel image and enhancing the color intensity of the protein spots. In the video, the gel is destained using a specific solution after staining, which helps to remove any unbound dye and improve the overall quality of the protein pattern on the gel.

Highlights

The video demonstrates the power of a modified Kumasi staining protocol for detecting proteins in polyacrylamide gels.

The example of two-dimensional gel electrophoresis is used to illustrate the procedure.

Immobilized pH gradient strips are rehydrated in denaturing buffer conditions for First Dimension separation.

Proteins are separated according to their isoelectric points during the focusing process.

Second dimension separation is performed by SDS-PAGE.

The Kumasi staining protocol is highlighted for its superior features in sensitivity, costs, and practicality.

Kumasi Brilliant Blue is a dye used for visualizing proteins after electrophoretic separations.

The protocol is especially useful in analytical proteomics for determining myocardial protein expression changes.

The first dimension of 2D gel electrophoresis is isoelectric focusing (IEF) using immobilized pH gradient strips.

Protein samples are applied via anodic cup loading during the focusing process.

The procedure involves re-swelling of the IPG strips and protein sample application.

The second dimension is performed by SDS-PAGE on a vertical electrophoresis system.

Colloidal Kumasi staining is prepared by sequential addition of components in a specific order.

The staining solution can be prepared in advance and stored in the dark until use.

Efficient washing of the gel is crucial to remove residual SDS for proper dye binding.

The 2D gel should be distinctly resolved and stained well with dark blue protein spots.

The Kumasi staining protocol is a good alternative to non-labeling methods of gel-based proteomics.

The procedure requires little labor and can detect as little as one nanogram of protein.

Transcripts

terrible

[Music]

in this video we will demonstrate the

power of a modified Kumasi staining

protocol for detecting several nanograms

of protein in polyacrylamide gels using

the example of two-dimensional gel

electrophoresis

prior to First Dimension separation

immobilized pH gradient strips are

rehydrated in denaturing buffer

conditions

after overnight re-swelling of the

strips protein samples are applied via

anodic cup loading during the focusing

process the proteins are separated

according to their isoelectric points

second dimension separation is performed

by SDS page

the gel is then transferred to a

staining dish and the proteins are

detected by staining with colloidal

Kumasi

hello I am Nadine dubala from the

laboratory of sabinemetska in the

biological Medical Research Center at

the University of Dusseldorf today I

will draw your attention to a widely

non-famous procedure according to kangan

colleagues Kumasi Brilliant Blue is a

Dye commonly used for the visualization

of proteins after electrophoretic

separations but is often assumed to be

less applicable for comparative

proteomics however kung's colloidal

staining offers Superior features

concerning sensitivity costs and

practicality we use Kung staining

protocol in our laboratory for nearly

every jail based application but

especially in analytical proteomics to

determine myocardial protein expression

changes I will demonstrate the use of

this method to detect myocardial

proteins in the alkaline pH range of

two-dimensional electrophoresis gels so

let's get started

the first dimension of 2D gel

electrophoresis is isoelectric focusing

or ief an immobilized pH gradient strips

or ipg strips are used for this analysis

prior to ief four ipg strips are

rehydrated individually in 125

microliters of rehydration solution

using the immobilin dry strip

re-swilling tray for at least 10 hours

when the ipg strips are rehydrated and

ready for protein sample application TCA

precipitated protein samples from four

different heart preparations are

dissolved in ief Sample buffer to a

concentration of 100 micrograms protein

per 100 microliters

solubilization of the protein samples

will take at least 30 minutes at room

temperature on a vortexer

in the meantime prepare the additional

components of the immobiline dry strip

kit to perform ief in the multi-4

electrophoresis unit

first set the temperature of the cooling

unit to 20 degrees Celsius

pipette three to four milliliters of

cover fluid onto the cooling plate and

position the immobilin dry strip tray

onto it with the red electrode

connection towards the anode

pour about 10 milliliters of cover oil

into the immobilene dry strip tray and

place the immobilin dry stripper liner

with the groove side up onto the oil

covered tray

next remove the rehydrated strip from

the re-swelling tray and transfer it

with gel side up to the grooves of the

aligner

the acidic end of the strip should face

towards the anode

at the end all strips should be aligned

with their anodic edges

Place distilled water saturated

electrode strips laterally across the

ends of the gel strips making sure that

the electrode strips at least partially

contact the gel surface

finally position the electrodes on the

electrode strips so that the colored

Mark is directed towards the

corresponding electronic contact

position the sample cup bar near the

anode at the immobilene dry strip tray

and place a sample cup above each ipg

strip gently press the sample cups down

to ensure good contact with the ipg

strip

pour 70 to 80 milliliters of cover oil

into the tray to cover the ipg strips no

oil should leak into the sample cups if

oil leaks into the cup remove it with a

pipette and adjust the cups again

finally add about 150 milliliters of

cover oil to completely cover the sample

cups

now apply the protein samples into the

sample cups by pipetting Under the

surface of the cover fluid

the volume of the cup is 100 microliters

once all protein samples have been

applied connect All Leads plug the

multi-4 unit to the power supply and

perform isoelectric focusing for 11.1

kilovolt hours in gradient mode

following ief the ipg strips are placed

into a tray with gel side up and

subjected to reduction and alkylation

during equilibration

for reduction use 2.5 milliliters of one

percent diethyothritol equilibration

solution per strip

after that shake for 15 minutes and then

completely replace the dtt solution and

alkylate the strips in 2.5 percent Iota

acetamide equilibration solution with

shaking for another 15 minutes

rinse the equilibrated strips once in a

water filled Beaker and blot them onto

filter paper to remove excess

equilibration buffer

next transfer the strips in 1X SDS

running buffer

the proteins in the strips are now ready

for the second dimension of the 2D gel

electrophoresis

the second dimension is performed by SDS

page on a vertical electrophoresis

system

an appropriate number of one millimeter

thick 12 percent acrylamide gels are

cast one day before use and kept covered

with wet paper at room temperature

the equilibrated ipg strips are placed

on the top of the separating gels and

fixed with hot agarose solution

proteins are separated for 2.5 hours

starting at 80 Volts for 15 minutes

followed by 120 volts until the die

front reaches the bottom of the gel

cassette

[Music]

while the proteins are being separated

by SDS page prepare the solutions for

colloidal Kumasi staining of the gel

when making the staining solution it is

very important to maintain the

sequential addition of the components in

the following order

first dissolve 100 grams of aluminum

sulfate in approximately 1.5 liters

millikub water in a beaker and stir

after the aluminum sulfate is dissolved

intermix 200 milliliters of ethanol and

add 0.4 grams CBD g250 to the solution

as soon as the CBB g250 is completely

dissolved add 47 milliliters of

orthophosphoric acid

this step allows the kamasi molecules to

aggregate into their colloidal state

finally add Millie Q water to a final

volume of 2 liters

the final standing solution will have

particles swimming around

do not filter this solution

the staining solution can also be

prepared in advance and stored in the

dark until use

when the SDS gel is completed carefully

remove the gel from the glass plates and

transfer it into a staining dish filled

with Millie Q water

place the dish with the gel on a

horizontal Shaker and shake for 10

minutes

repeat the wash two more times for about

five to ten minutes with fresh Milli

Cube water

it is important to wash the gel well

because remaining SDS on the gel can

disrupt The Binding of the dye to the

protein and cause pore sensitivity

at the end of the third wash pour out

the water from the dish

the gel is now ready to be stained

first shake the Kumasi solution to

disperse the colloidal particles evenly

then add enough solution to cover the

gel

place the dish with the Kumasi solution

covered gel on a Shaker and agitate for

2 to 12 hours

after 10 minutes you will see the first

protein spots appearing 80 to 100

percent of the staining can be

accomplished within two hours of

incubation but we recommend an overnight

incubation to achieve close to 100

staining

when staining is complete remove the

Kumasi solution and rinse the gel twice

with Millie Q water

[Music]

after rinsing the gel remove sticking

dye particles from the staining dish

with a lint-free paper towel

add destaining solution to the gel and

destain for 10 to 60 Minutes on the

shaker

finally rinse the gel twice with Millie

Q water the gel will resume its original

thickness and the color intensity of the

Kumasi stain will also be enhanced

foreign

if you follow this protocol your 2D gel

should be distinctly resolved and

stained well with dark blue protein

spots the result of our Kumasi staining

of 2D gels is comparable to that of

silver staining according to the

protocol of chefchenko at all which is

reported to be of high sensitivity and

compatibility with Mass spectrometry

[Music]

using the example of two-dimensional gel

electrophoresis we have just shown you

how fast simple and sensitive cancers

staining is to detect proteins in

polyacrylamide shares sustaining can be

completed within two hours the procedure

itself requires little labor and last

but not least you might be able to

detect as little as one nanogram of

protein

so this protocol is definitely a good

alternative to the non-labeling methods

of JailBase proteomics

when you use this staining procedure

it's important to remember to use

analytical grade chemicals and water

fires Purity furthermore the sequential

addition of the components to the

staining solution is the critical step

in the preparation

finally don't forget to efficiently wash

the residual SDS from the jails after

the electrophoretic separation otherwise

you won't see anything on your juice so

that's it thanks for watching and good

luck with your experiments