If you are working in the field of biology then you know how important the use of citrate buffer is. It has many uses in the lab and is often found in stock alongside solutions such as PBS.
In this article I will discuss everything to know about reusing citrate buffer to ensure you have the best results as well as some related questions, such as temperature and storage.
As generally accepted in the scientific community, you should not reuse citrate buffer. It is not worth it to reuse citrate buffer because it is not expensive to make and you do not know exactly when it has lost its effectiveness. Your tissue should be getting a fresh, potent solution because tissue is limited, whereas reagents are not.
Many scientifically related fields call for the use of a citrate buffer at one point or another. This makes sense, however, as a citrate buffer is an important and useful chemical solution that can be used to do many different jobs.
What does a citrate buffer do?
As previously stated, using a citrate buffer is a common requirement if you’re working in biology, chemistry or any other scientifically related field of work. Some of its uses include RNA isolation as it can prevent base hydrolysis, immunofluorescence as it can help bring out the immunostaining, and antigen detection, as it breaks the cross links between antigens and other substances.
Antigen Retrieval
One main job that a citrate buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0) can be used for is antigen retrieval. Antigen retrieval is a process where an antibody is manipulated to target a specific protein inside a selected segment of tissue. The result of a properly carried out procedure of antigen retrieval will recover hidden epitopes.
Antigen retrieval can be a lifesaver for scientists during many different experiments. It’s common to follow the practice of preserving tissues with formaldehyde. However, formaldehyde fixation is known to cross link amino acid residues. As a result tissue specimens hide strains of protein, which these scientists were hoping to find.
Function In Antigen Retrieval
By carrying out a quick antigen retrieval process, any selected tissue specimen can easily have this chemical mishap erased in no time. As a result, the preserved selected tissue specimen can be used again as you will be able to retrieve proteins that you need from it once again.
Although there are a few ways that you can do this, by far the best, easiest, and most effective way to carry out an antigen retrieval process is by using a citrate buffer. This is because a citrate buffer can easily unlatch any cross-links that have been formed between antigens and any other substances in a selected tissue specimen.
Microwaving
To use a citrate buffer safely and effectively you need to prepare your citrate buffer properly. One key element that you need to pay attention to when preparing your citrate buffer for any experiment is the temperature at which you will use your citrate buffer.
Microwaving citrate buffer is used for heat induced epitope retrieval and it is commonly done at near boiling temperature for up to 20 minutes. You need to get your citrate buffer to the right temperature .
There are many ways that you can get your citrate buffer at the right temperature for your scientific experiment. Some ways in which you can get your citrate buffer at the right temperature are as follows;
-Using a pressure cooker
-Using a microwave
-Using a hot water bath
-Using a steamer
However, although there are many ways for you to get your citrate buffer at the right temperature, the easiest, best, and quickest way to do so is by using a microwave.
Here is how you safely and effectively microwave your citrate buffer to the temperature that you need it to be. Since the process of antigen retrieval is one of the most common uses of a citrate buffer, I will use it as an example.
- Deparaffinize your selected specimens of tissue. You need to deparaffinized your selected specimens of tissue with xylene. Soak your selected specimens of tissue for 5 minutes on each side.
- After this, you must soak your selected specimens of tissue in 100% ethanol. Soak your selected specimens of tissue in 100% ethanol for three minutes.
- After this, you must soak your selected specimens of tissue in 95% ethanol. Soak your selected specimens of tissue in 95% ethanol for one minute.
- After this, you must soak your selected specimens of tissue in 90% ethanol. Soak your selected specimens of tissue in 90% ethanol for one minute.
- After this, you must soak your selected specimens of tissue in 80% ethanol. Soak your selected specimens of tissue in 80% ethanol for one minute then rinse clean with water.
- While this procedure with your selected specimens of tissue is being carried out, you can place your citrate buffer in a staining dish and microwave it to the appropriate temperature. The temperature that you want your citrate buffer to be at for antigen retrieval is 95-100 degrees celsius. For you not to overheat or underheat your citrate buffer, it’s recommended by experts to do your heating in fifteen to twenty-minute intervals so that you can check constantly and consistently.
- When the previous steps have been completed, you can combine your selected specimens of tissue with your citrate buffer on the staining dish. Then proceed to incubate your staining dish in a secure location for twenty to forty minutes for the best results while keeping the temperature at the correct level.
Always be very careful when heating chemicals to such high temperatures, especially when the pH is not neutral. If the solution starts boiling, you can get bubbles and splashes, in addition to glass containers possibly breaking from the heat and the hot contents splattering on you.
If you want to learn more about preparing citrate buffer, you can check out this video:
Storage
Fresh or leftover citrate buffer needs to be stored in the right fashion to keep its effectiveness. This is how you need to store your citrate buffer the right way so that you can resume it for future projects.
Temperature
Just as you must use your citrate buffer at the right temperature it is equally, if not more, important that you store your leftover citrate buffer at the right temperature as well. The right temperature to store your leftover citrate buffer is at room temperature for up to 3 months or at 4C for longer storage.
Container
Another important thing to consider when you want to store your leftover citrate buffer for a future project of yours that will call for the use of a citrate buffer is what container you store your citrate buffer in. You must store your citrate buffer in its original container. However, if the original container that your citrate buffer came in is no longer in prime condition, then you must store your citrate buffer in a different container. It’s recommended that this new container be as close to the original container in its makeup as possible. Make sure that this new container is made of the right materials (glass is preferred over plastic) and can be effectively and tightly sealed.
I hope this article answered some of your questions about citrate buffer and reusing it. Click the following link to learn how to make microscope mounting medium.