In this video, we will demonstrate the power of a modified kumasi staining protocol for detecting several nanograms of protein in poly acrylamide 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 de Bala from the laboratory of Sabina Meka in the biological medical research center at the University of Olor. Today I will draw your attention to a widely non-famous procedure according to Kangan colleagues, Cy Brilliant Blue is a dyke commonly used for the visualization of proteins after electrophoretic separations, but is often assumed to be less applicable for comparative proteomics.
However, cos colloidal staining offers superior features concerning sensitivity costs and practicality. We use K staining protocol in our laboratory for nearly average gel 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. Got it. The first dimension of 2D gel electrophoresis is isoelectric focusing or IEF and 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 IM Mobilly dry strip swelling 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 vortex.
In the meantime, prepare the additional components of the IM Molene dry strip kit to perform IEF in the multi four 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 Immobilien dry strip tray onto it with the red electrode connection towards the anode. Pour about 10 milliliters of cover oil into the Immobilien dry strip tray and place the Immobilien dry strip aligner 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 immobilien 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 four 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 1%DI three et all equilibration solution per strip. After that shake for 15 minutes and then completely replace the DTT solution and alkylate the strips in 2.5%I oto acetamide equilibration solution with shaking for another 15 minutes. Rinse the equilibrating strips once in a water-filled beaker and blot them onto filter paper to remove excess equilibration buffer.
Next, transfer the strips in one XSDS 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%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 agro solution per 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.
While the proteins are being separated by SDS page, prepare the solutions for colloidal kumasi staining at 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 aluminium sulfate and approximately 1.5 liters milli cube water in a beaker and stir After the aluminium sulfate is dissolved, intermixed 200 milliliters of ethanol and add 0.4 grams CBG two 50 to the solution.
As soon as the CBB G two 50 is completely dissolved, add 47 milliliters of ortho phosphoric acid. This step allows the kamasi molecules to aggregate into their colloidal state. Finally, add milli Q water to a final volume of two 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 Milli 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 10 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 poor 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 two to 12 hours. After 10 minutes, you'll see the first protein spots appearing.
80 to a hundred 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 Kamasi solution and rinse the gel twice with Milli Q water.
After rinsing the gel, remove sticking dye particles from the staining dish with a lint-free paper towel. Add des staining solution to the gel and detain for 10 to 60 minutes on the shaker. Finally, rinse the gel twice with Milli Q water.
The gel will resume its original thickness and the color intensity of the kumasi stain will also be enhanced. 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 Chef Chenko et al, which is reported to be of high sensitivity and compatibility with mass spectrometry. Using the example of two dimensional gel electrophoresis, we have just shown you how fast, simple and sensitive kangs cooma sustaining is. To detect proteins in po acrylamide gels 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 unlabeling methods of jail based proteomics.
When you use sustaining procedure, it's important to remember to use analytical grad chemicals and water of highest purity. Furthermore, the sequential addition of the components to the staining solution is a critical step in the preparation. Finally, don't forget to efficiently wash the residual SDES from the gels after the electrophoretic separation.
Otherwise, you won't see anything on your gels. So that's it. Thanks for watching and good luck with your experiments.
