Citrate Buffer recipe
Introduction
The authorship is often contributed to Gomori (1955) but Gomori is in this publication citing an article that is seven years older (Lillie 1948) as original source. Citrate buffer is a universal buffer used in various reactions. It is sometimes combined with sodium citrate or completely replaced by sodium citrate buffer. However, alkalized citrate buffer has a similar composition to sodium citrate buffer.
Applications
This buffer is commonly used in different fields of biochemistry as a basic pH buffer with adjustable pH for enzymatic solutions and chromatography. In the era of molecular biology, the citrate buffer was found to be useful in
- RNA isolation because it prevents the bases from hydrolysis,
- heat-induced antigen retrieval protocols since it breaks the cross-links and in DNA fluorescent in situ hybridization.
Composition
There are several ways of preparation of this buffer.
| Reagent | Molecular weight | 1X molarity | Add for 500ml of 1X | Add for 1L of 1X |
|---|---|---|---|---|
| Citric acid | 192.1 | 100 mM | 9.6 g | 19.2 g |
| Dihydrate tri-sodium citrate | 258.1 | 100 mM | 12.9 g | 25.8 g |
Table 1.
*Tween is polyethylene glycol sorbitan monolaurate. It is used as a detergent in this solution.
| Reagent | Molecular weight | 1X molarity | Add for 500ml of 1X | Add for 1L of 1X |
|---|---|---|---|---|
| Citric acid | 192.1 | 100 mM | 9.6 g | 19.2 g |
| Dihydrate tri-sodium citrate | 258.1 | 100 mM | 12.9 g | 25.8 g |
Table 2.
*Tween is polyethylene glycol sorbitan monolaurate. It is used as a detergent in this solution.
pH
For enzymes: The pH can be adjusted from 3,0 to 6,2 according to the specific needs of your enzyme by changing the composition or by adding HCl to acidify and NaOH to alkalize the buffer. However, the usage of strong acids and hydroxides can change the ionic strength, and therefore, it could affect the experiment.
For antigen retrieval: The pH 6,0 is used, adjusted by NaOH.
Preparation
For antigen retrieval:
- Add about ¾ of the final volume of distilled water to the glass beaker.
- Prepare the exact amounts of citric acid using balance and put it into the water to dissolve. A magnetic stirrer can be used to speed up the process.
- After the citric acid is dissolved, add distilled water to the final volume.
- Adjust the pH to 6 with 0,1 M NaOH.
- Add 0,5 ml of Tween and mix really well.
For enzymes, 1 l of the final solution:
- First, prepare the stock solutions of citric acid and sodium citrate:
- Add about ¾ of the final volume of distilled water to the glass beaker.
- Prepare the exact amounts of citric acid/sodium citrate using balance and put it into the water to dissolve. A magnetic stirrer can be used to speed up the process.
- After the citric acid is dissolved, add distilled water to the final volume.
- In the next step, mix specific amounts of citric acid solution and sodium citrate solution according to the table below to obtain the buffer of the required pH.
| Required pH | Amounts of stock solutions (ml) | ||
|---|---|---|---|
| Required pH | Amounts of stock solutions (ml) | ||
| Required pH | 0.1 M citric acid | 0.1 M sodium citrate | |
| 3 | 930 | 70 | |
| 3.2 | 874 | 126 | |
| 3.4 | 800 | 200 | |
| 3.6 | 740 | 260 | |
| 3.8 | 700 | 300 | |
| 4 | 660 | 340 | |
| 4.2 | 630 | 370 | |
| 4.4 | 560 | 440 | |
| 4.6 | 510 | 490 | |
| 4.8 | 460 | 540 | |
| 5 | 410 | 590 | |
| 5.2 | 360 | 640 | |
| 5.4 | 320 | 680 | |
| 5.6 | 274 | 726 | |
| 5.8 | 236 | 764 | |
| 6 | 190 | 830 | |
| 6.2 | 144 | 856 | |
Table 3.
Storage
It can be stored at room temperature for 3 months. At a lower temperature (4 °C), the time could be prolonged but the solution should not freeze to avoid precipitation.
Points to be noted
- Tween is an irritant so it should not come in contact with skin or mucosae and it should not be swallowed. It is also critical to mix it well with water when preparing the buffer.
- Citric acid is a weak acid but it can cause eye irritation.