In a previous video titled Electrophoretic Separation of Proteins, we demonstrated how a complex mixture of proteins could be separated using gel electrophoresis. In this video, we will show you four different gel staining methods for visualizing the location of the proteins in the gel. Hi, I'm Sean Gallagher from UVP working with the Proteomics Center here at the KEG Graduate Institute.
Today I'll be showing you a number of techniques for visualizing proteins after separation by electrophoresis. This includes Kamasi blue silver staining, cyro orange and cyro ruby. So let's get started with Kamasi blue First.
The detection of protein bans in a gel by Kamasi blue staining depends on non-specific binding of a dye. In this case, Kamasi brilliant blue G two 50. In this method, proteins separated in a poly acrylamide gel are washed using high purity water.
The location of the proteins is then detected using a water-based kamasi blue stain. The detection limit is 0.3 to one microgram per protein band. To begin, start with a poly acrylamide gel in which proteins have been separated by electrophoresis.
While agitating slowly place the poly acrylamide gel in a plastic container filled with 300 milliliters of high purity water. Leave for five minutes. Discard and repeat two more times after the third wash.
Pour out the water and cover the gel with Kamasi blue staining solution for one hour while agitating slowly after one hour of staining, pour out the staining solution. Rinse the gel briefly by placing in about 200 milliliters of water for 30 minutes. At this point, the gel may be stored in water for future imaging.
The gel may also be dried at this stage to maintain a permanent gel record. To do this, place the gel on two sheets of Watman three mm filter paper and cover the top with plastic wrap. Then dry in a conventional gel dryer for one to two hours at about 80 degrees celsius.
Another protein staining method is by silver staining, which we will demonstrate next. Although the Kamasi blue staining method is easier and more rapid, silver staining is considerably more sensitive with a detection limit of 0.3 nanograms BSA. The detection of protein bands in a gel by silver staining depends on the binding of silver to various chemical groups in the protein.
The following protocol is based on the Silver Quest silver staining kit from in Vitrogen. The protocol is derived from the fast staining protocol, approximately 30 minutes and represents one of the many kit based chemistries used for silver staining compatible with mass spectrometry. In this case, we'll be using an in vitrogen precast mini gel before beginning silver staining.
Make sure to wear gloves at all times to avoid fingerprint contamination. Place the gel in a microwave safe tray with 100 milliliters reagent grade water. Briefly rinse and then pour off the water.
Next, add 100 milliliters of fixative, composed of 40%ethanol, 10%acetic acid in high purity water and microwave for 30 seconds. The microwave process shortens the necessary incubation steps by accelerating the binding and staining. At this point, place the gel on a shaker and slowly rock for five minutes.
After five minutes, pour off the fixative, add 100 milliliters, 30%ethanol to wash the gel microwave for 30 seconds, and then place the gel on a shaker for five minutes. After five minutes, pour off the ethanol. Now add 100 milliliters of sensitizing reagent microwave for 30 seconds, and then place on a shaker for two minutes.
After the two minutes, pour off the sensitizing reagent. Add 100 milliliters reagent grade water microwave for 30 seconds, and place on a shaker for two minutes. Pour off and repeat for a total of two wash cycles.
Now add 100 milliliters of staining solution, microwave for 30 seconds and slowly agitate for five minutes. After the five minutes, pour off the staining solution. Add 100 milliliters reagent grade water, skip the microwave, step and place directly on a shaker for 20 to 60 seconds.
Discard the water and immediately add 100 milliliters of the developing solution and develop until protein bands become visible. With the background remaining clear, this will take approximately five to 10 minutes. At this point, add 10 milliliters of stopper solution to the developer to arrest the development for best results.
Photograph the gel as soon as possible as there may be slight changes in color intensity and increases in non-specific background elements. Over time, the gel you are seeing contains residual proteins After electro blotting, dry the gel to maintain a permanent record or store in a sealable plastic bag. Next, we demonstrate a method for fluorescently staining gels.
Fluorescent dyes have a number of advantages over traditional protein stains. Cipro orange protein gel stains, which we will outline here can detect one to two nanograms of protein per band. This is more sensitive than kamasi brilliant blue staining and as sensitive as many silver staining techniques.
Fluorescent staining is straightforward with less hands on time than typical silver staining protocols and is complete in under one hour. Stained proteins can be visualized using a standard 300 nanometer UV transluminator or a laser scanner. In this case, we will again be using an in vitrogen precast mini gel to begin prepare and separate proteins using SDS page.
But use 0.05%SDS in the running buffer instead of the usual 0.1%SDS in order to reduce background fluorescence. To begin the staining procedure, pour cipro orange fluorescent staining solution into a small plastic dish for one or two standard sized mini gels. Use about 50 milliliters of staining solution.
It is important to note that staining dishes should be cleaned and rinsed well before use as any detergents will interfere with the staining. Now, place the gel into the staining solution. Cover the container with aluminum foil to protect the dye from bright light.
Gently agitate the gel at room temperature for 40 to 60 minutes. Discard the staining solution and then rinse the gel for less than one minute with 7.5%acetic acid, about 100 milliliters to remove the excess stain from the gel surface, detain the gel by incubating overnight in 0.1%tween 20 to visualize and photograph protein bands. Place the fluorescently labeled gel directly on a standard 300 nanometer transluminator or a blue light transluminator.
Next, we will demonstrate an alternative fluorescent staining method, which uses Cipro ruby fluorescent dye. The Cipro ruby staining method was designed especially for use in two dimensional page, but has proven to be the most sensitive protein gel stain for standard one dimensional SDS page. To begin transfer the gel to a clean polypropylene or PVC dish of appropriate size.
Incubate the gel in the appropriate amount of undiluted cipro ruby protein gel stain with continuous gentle agitation on an orbital shaker at 50 RPMs. Cover the gel and incubate for at least three hours. Transfer the gel to a clean staining dish and wash it once in a 10%methanol, 7%acetic acid solution, recover and place the gel back on the rocker for 30 minutes.
In order to reduce background fluorescence and increase sensitivity, the gel is now ready to be viewed and photographed. I want to thank you for watching today's protocols today. We've shown you a number of different procedures for visualizing proteins after electrophoresis.
Some are very sensitive, such as the fluorescent staining and silver staining, and some are routine such as kamasi blue staining. It's important to remember that reagent purity is critical. Using ultra pure water is also very important, and also the timing on each one of the steps.
You need to pay close attention to that in order to get reproducible results also. That's it and good luck in your next set of experiments.
