Preparation of Different pH Buffer

pH Buffer solution

• A pH buffer or hydrogen ion buffer is defined as an aqueous solution that is composed of a mixture of a weak acid and its conjugate base, or vice versa.
• In addition of a small amount of strong acid or base, it shows very little changes in pH.
• pH buffer is mainly used to maintain the pH value constant in different chemical applications.
• For pH regulation in nature, different types of systems are used such as bicarbonate buffering system is used to regulate the pH of blood, and bicarbonate also acts as a buffer in the ocean.
• In a biological system, the pH buffer helps in maintaining the proper function of enzymes.

There are present different types of pH buffer such as;

ACES Buffer (0.1 M, 6.7 pH) Preparation 

ACES buffer is one of the Good’s zwitterionic buffer with a pH range of 6.1-7.2. To prepare 1L of ACES Buffer (0.1 M, 6.7 pH) the following components are required;

Component	Amount	Concentration
ACES (mw: 182.20 g/mol)	18.22 g	0.1 M

ACES Buffer (0.1 M, 6.7 pH) Preparation Steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 18.22 g of ACES to the solution.
3. Adjust solution to desired pH by 10N NaOH.
4. Add dH2O until volume is 1 L.

Acetate Buffer (0.1 M, pH 5.0) Preparation 

Sodium acetate buffers are used for purification and precipitation of nucleic acids, as well as for protein crystallization and staining gels used in protein electrophoresis. To prepare 1L of Acetate Buffer (0.1 M, pH 5.0) the following components are required;

Component	Amount	Concentration
Sodium Acetate (mw: 82 g/mol)	5.772 g	0.07 M
Acetic Acid (mw: 60.05 g/mol)	1.778 g	0.03 M

Acetate Buffer (0.1 M, pH 5.0) Preparation Steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 5.772 g of Sodium Acetate to the solution.
3. Add 1.778 g of Acetic Acid to the solution.
4. Adjust solution to desired pH using 10N HCl (typically pH ≈ 5.0).
5. Add distilled water until volume is 1 L.

Acetate Buffer (pH 3.6 to 5.6) Preparation

Sodium acetate buffers are used for purification and precipitation of nucleic acids, as well as for protein crystallization and staining gels used in protein electrophoresis. It is very popular in hematology, since there is some evidence that acetate-buffered infusions show improved stability. Acetate buffers are inexpensive and simple to prepare, and can be stored at room temperature.

To prepare 1L of Acetate Buffer (pH 3.6 to 5.6) the following components are required;

Component	Amount	Concentration
Sodium Acetate (mw: 82.03 g/mol)	7.721 g	0.0941 M
Acetic Acid (mw: 60.05 g/mol)	353 mg	0.0059 M

Acetate Buffer (pH 3.6 to 5.6) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 7.721 g of Sodium Acetate to the solution.
3. Add 0.353 g of Acetic Acid to the solution.
4. Adjust solution to final desired pH using HCl or NaOH
5. Add distilled water until volume is 1 L.

ADA Buffer (0.5 M, 6.6 pH) Preparation

ADA buffer is one of the Good’s buffer with a pH range of 6.0-7.2. It is often used in cell culture media as a chelating agent for H+, Ca2+, and Mg2+. To prepare 1L of ADA Buffer (0.5 M, 6.6 pH) the following components are required;

Component	Amount	Concentration
ADA (mw: 190.22 g/mol)	95.11 g	0.5 M

ADA Buffer (0.5 M, 6.6 pH) Preparation Steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 95.11 g of ADA to the solution.
3. Add dH2O until volume is 1 L.
4. ADA will not fully dissolve until the pH is raised. Adjust solution to desired pH by 10N NaOH.

Ammonium Acetate Preparation

Ammonium acetate is widely used in molecular biology in applications such as protein purification and precipitation. To prepare 1L of Ammonium Acetate the following components are required;

Component	Amount	Concentration
ammonium acetate (mw: 77.08 g/mol)	770 g	10 M

Ammonium Acetate Preparation Steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 770 g of ammonium acetate to the solution.
3. Add distilled water until volume is 1 L.
4. Sterilize the solution by passing it through a 0.22-µm filter. Store the solution in tightly sealed bottles at 4°C or at room temperature. Ammonium acetate decomposes in hot H2O and solutions containing it should not be autoclaved.

Ammonium Bicarbonate (50 mM, pH 7.8) Preparation

To prepare 1L of Ammonium Bicarbonate (50 mM, pH 7.8) the following components are required;

Component	Amount	Concentration
NH4HCO3 (mw: 79.06 g/mol)	4 g	0.0506 M

Ammonium Bicarbonate (50 mM, pH 7.8) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 4 g of NH4HCO3 to the solution.
3. Add distilled water until volume is 1 L.

Ammonium Sulfate, Saturated Preparation

Ammonium Sulfate is commonly used for protein precipitation and fractionation due to enhancing hydrophobic interactions. To prepare 1L of Ammonium Sulfate, Saturated the following components are required;

Component	Amount	Concentration
ammonium sulfate (mw: 132.14 g/mol)	761 g	5.759 M

Ammonium Sulfate, Saturated Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 761 g of ammonium sulfate to the solution.
3. Add distilled water until volume is 1 L.
4. Dissolve with stirring and gentle heating. Cool to room temperature and adjust the pH to 7 (with NH4OH or an acid such as HCl). If excess is to be stored, sterilize the solution by filtering through a 0.2-µm filter and store at 4°C.

BES-Buffered Saline (2X) (0.05 M, pH 6.95) Preparation

BES-buffered saline is often used in the calcium phosphate-mediated transformation of mammalian cells. To prepare 1L of BES-Buffered Saline (2X) (0.05 M, pH 6.95) the following components are required;

Component	Amount	Concentration
BES (mw: 213.25 g/mol)	10.66 g	0.05 M
NaCl (mw: 58.44 g/mol)	16.36 g	0.28 M
Na2HPO4 (mw: 141.96 g/mol)	210 mg	0.0015 M

BES-Buffered Saline (2X) (0.05 M, pH 6.95) Preparation Steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 10.66 g of BES to the solution.
3. Add 16.36 g of NaCl to the solution.
4. Add 210 mg of Na₂HPO₄ to the solution.
5. Adjust solution to desired pH using 1M NaOH (typical pH = 6.95)
6. Add dH2O until volume is 1 L.

Bicine (1 M, pH 8.26) Preparation 

Bicine is used in peptide and protein biochemical applications, and is ideal for low temperatures. Bicine is a Zwitterionic compound, also known as a dipolar ion. To prepare 1L of BES-Buffered Saline (2X) (0.05 M, pH 6.95) the following components are required;

Component	Amount	Concentration
Bicine (mw: 163.17 g/mol)	163.17 g	1 M

Bicine (1 M, pH 8.26) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 163.17 g of Bicine to the solution.
3. Adjust solution to desired pH with 10N NaOH.
4. Add dH2O until volume is 1 L.

Bis-Tris Buffer (1 M, 6.5 pH) Preparation 

Bis-Tris buffer is used in molecular biology for applications involving both proteins and nucleic acids. Similar to tris-buffers, it has good buffer capacity and can be stored at room temperature. Bis-Tris interacts with several metal ions including Cu and Pb. To prepare 1L of Bis-Tris Buffer (1 M, 6.5 pH) the following components are required;

Component	Amount	Concentration
Bis-Tris (mw: 209.24 g/mol)	209.24 g	1 M

Bis-Tris Buffer (1 M, 6.5 pH) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 209.24 g of Bis-Tris to the solution.
3. Starting pH without HCl is 9.90. Concentrated HCl is used to adjust the pH. Filter sterilize or autoclave before use or storage.
4. Add dH2O until volume is 1 L.

Borate Buffer Preparation

Borate buffer is a widely use buffer with many applications. Borate buffers that contain sodium chloride have been used to study corrosion. To prepare 1L of Borate Buffer the following components are required;

Component	Amount	Concentration
Boric acid (mw: 61.83 g/mol)	6.185 g	0.1 M
NaCl (mw: 58.44 g/mol)	4.385 g	0.075 M
Sodium tetraborate (mw: 201.22 g/mol)	5.031 g	0.025 M

Borate Buffer Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 6.185 g of Boric acid to the solution.
3. Add 4.385 g of NaCl to the solution.
4. Add 5.031 g of Sodium tetraborate to the solution.
5. Add distilled water until volume is 1 L.
6. Use a stir bar and low heat to ensure that the boric acid dissolves fully. Sterilize by vacuum-filtration with a 0.22-µm filter. Adjust the pH to 8.4.

CAPS (0.5 M, pH 10.4) Preparation

CAPS is used commonly for Western Blot applications and other protein sorting procedures. The very high pH of this buffer allows for identification and sequencing of peptides and proteins with high isoelectric points (pl or IEP). To prepare 1L of CAPS (0.5 M, pH 10.4) the following components are required;

Component	Amount	Concentration
CAPS (mw: 221.32 g/mol)	110.66 g	0.5 M

CAPS (0.5 M, pH 10.4) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 110.66 g of CAPS to the solution.
3. Adjust solution to desired pH using 10N NaOH.
4. Add dH2O until volume is 1 L.

Carbonate-Bicarbonate Buffer (pH 9.2 to 10.6) Preparation

Commonly used for various immunoassay applications and for many protein and antibody conjugation procedures, including sandwich ELISA, which require experimental surface coatings. Carbonate-bicarbonate buffer is used extensively in molecular and cell biology, biochemistry, and in the medical field, where it is the most commonly used small intestine buffer. It has good buffering capacity and is easy to prepare, with excellent shelf life.

To prepare 1L of Carbonate-Bicarbonate Buffer (pH 9.2 to 10.6) the following components are required;

Component	Amount	Concentration
Sodium bicarbonate (mw: 84.01 g/mol)	1.05 g	0.0125 M
Sodium carbonate (anhydrous) (mw: 105.99 g/mol)	9.274 g	0.0875 M

Carbonate-Bicarbonate Buffer (pH 9.2 to 10.6) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 1.05 g of Sodium bicarbonate to the solution.
3. Add 9.274 g of Sodium carbonate (anhydrous) to the solution.
4. Add distilled water until volume is 1 L.

CHES (0.5 M, pH 9.5) Preparation

CHES is a dipolar ion (Zwitterionic) used at relatively high pH levels up to 10. It is employed in several biological research fields including enzymology, where it is useful for studying reactions above physiological pH range. Commonly used for electrophoresis, it has low reactivity with most metal ions.

To prepare 1L of CHES (0.5 M, pH 9.5) the following components are required;

Component	Amount	Concentration
CHES (mw: 207.29 g/mol)	103.65 g	0.5 M

CHES (0.5 M, pH 9.5) Preparation and Recipe steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 103.65 g of CHES to the solution.
3. Adjust solution to desired pH with 10N NaOH.
4. Add dH2O until volume is 1 L.

Citrate Buffer (0.1 M, pH 6.0) Preparation 

Citrate buffers can be used for RNA isolation, thanks to its ability to prevent base hydrolysis. It also finds use in antigen detection by breaking cross-links between antigens and any substances in its fixation medium.

To prepare 1L of Citrate Buffer (0.1 M, pH 6.0) the following components are required;

Component	Amount	Concentration
Sodium Citrate dihydrate (mw: 294.1 g/mol)	24.269 g	0.0825 M
Citric Acid (mw: 192.1 g/mol)	3.358 g	0.0175 M

Citrate Buffer (0.1 M, pH 6.0) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 24.269 g of Sodium Citrate dihydrate to the solution.
3. Add 3.358 g of Citric Acid to the solution.
4. Adjust solution to desired pH using 0.1N HCl (typically pH ≈ 6.0).
5. Add distilled water until volume is 1 L.

Citrate Buffer (pH 3.0 to 6.2) Preparation

Citrate buffers can be used for RNA isolation, due to its ability to prevent base hydrolysis. The buffer is also used for antigen detection by breaking cross-links between antigens and any substances in its fixation medium. Citrate buffer is a popular choice for immunofluorescent staining applications, as it can aid in stain intensity without contributing to background signal. It is a popular choice for biological research and clinical applications, including pathology, and has had an increasing role in the medical field, as it has been shown to mildly improve dialysis efficiency. It has a shelf life of up to 3 months at room temperature. 

To prepare 1L of Citrate Buffer (pH 3.0 to 6.2) the following components are required;

Component	Amount	Concentration
Sodium Citrate dihydrate (mw: 294.1 g/mol)	25.703 g	0.0874 M
Citric Acid (mw: 192.1 g/mol)	2.421 g	0.0126 M

Citrate Buffer (pH 3.0 to 6.2) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 25.703 g of Sodium Citrate dihydrate to the solution.
3. Add 2.421 g of Citric Acid to the solution.
4. Adjust solution to final desired pH using HCl or NaOH
5. Add distilled water until volume is 1 L.

Citrate-Phosphate Buffer (0.15 M, pH 5.0) Preparation

A traditional buffer originally introduced in 1921. Since the Citrate-Phosphate buffer (also known as the McIlvaine buffer) only has 2 ingredients, the recipe can be adjusted to a pH range of 3-8. It is used for multiple applications in cell biology, molecular biology, and hematology.

To prepare 1L of Citrate-Phosphate Buffer (0.15 M, pH 5.0) the following components are required;

Component	Amount	Concentration
Na2HPO4-2H2O (mw: 177.99 g/mol)	18.15 g	0.102 M
Citric acid (mw: 192.1 g/mol)	9.605 g	0.05 M

Citrate-Phosphate Buffer (0.15 M, pH 5.0) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 18.15 g of Na₂HPO₄-2H₂O to the solution.
3. Add 9.605 g of Citric acid to the solution.
4. Adjust solution to final desired pH using HCl or NaOH
5. Add distilled water until volume is 1 L.

Citrate-Phosphate Buffer (20 mM, pH 5.6) Preparation

A traditional buffer originally introduced in 1921. Since the Citrate-Phosphate buffer (also known as the McIlvaine buffer) only has 2 ingredients, the recipe can be adjusted to a pH range of 3-8. It is used for multiple applications in cell biology, molecular biology, and hematology.

To prepare 1L of Citrate-Phosphate Buffer (20 mM, pH 5.6) the following components are required;

Component	Amount	Concentration
Na2HPO4 (anhydrous) (mw: 141.96 g/mol)	2.82 g	0.0199 M
Citric acid (mw: 192.12 g/mol)	4.2 g	0.0219 M

Citrate-Phosphate Buffer (20 mM, pH 5.6) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 2.82 g of Na2HPO4 (anhydrous) to the solution.
3. Add 4.2 g of Citric acid to the solution.
4. Add distilled water until volume is 1 L.
5. Sterilize by autoclaving. (The final solution contains 20 mM Na2HPO4.)

Citrate-Phosphate Buffer (50 mM, pH 5.6) Preparation

A traditional buffer originally introduced in 1921. Since the Citrate-Phosphate buffer (also known as the McIlvaine buffer) only has 2 ingredients, the recipe can be adjusted to a pH range of 3-8. It is used for multiple applications in cell biology, molecular biology, and hematology.

To prepare 1L of Citrate-Phosphate Buffer (50 mM, pH 5.6) the following components are required;

Component	Amount	Concentration
Na2HPO4 (anhydrous) (mw: 141.96 g/mol)	7.1 g	0.05 M
Citric acid (mw: 192.12 g/mol)	11.5 g	0.0599 M

Citrate-Phosphate Buffer (50 mM, pH 5.6) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 7.1 g of Na2HPO4 (anhydrous) to the solution.
3. Add 11.5 g of Citric acid to the solution.
4. Add distilled water until volume is 1 L.
5. Sterilize by autoclaving. (The final solution contains 50 mM Na2HPO4.)

Diethanolamine (1 M, pH 9.8) Preparation

Diethanolamine (DEA) is the buffer of choice in applications employing concentrated Tropix substrates for the detection of alkaline phosphatase. DEA buffers provide favorable reaction kinetics and greater sensitivity compared to most buffer systems.

To prepare 1L of Diethanolamine (1 M, pH 9.8) the following components are required;

Component	Amount	Concentration
diethanolamine (mw: 105.14 g/mol)	105.14 g	1 M
MgCl2-6H2O (mw: 203.30 g/mol)	100 mg	0.0005 M

Diethanolamine (1 M, pH 9.8) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 105.14 g of diethanolamine to the solution.
3. Add 100 mg of MgCl₂-6H₂O to the solution.
4. Adjust the pH to 9.8 with 10 M HCl solution.
5. Add dH2O until volume is 1 L.

EBSS (magnesium, calcium, phenol red) (pH 7.0) Preparation

EBSS is the abbreviation of Earle’s Balanced Salt Solution, and is in common use for cell culture applications including washing and dilution in CO2 environments. The addition of the magnesium and calcium ions, along with phenol red, is to assist in reagent preparation. Phenol red is a weak estrogen and should not be used long-term with responsive cell cultures. For other buffers containing Phenol Red, click here.

To prepare 1L of EBSS (magnesium, calcium, phenol red) (pH 7.0) the following components are required;

Component	Amount	Concentration
CaCl2 (mw: 110.98 g/mol)	200 mg	0.001 M
MgSO4-7H2O (mw: 246.47 g/mol)	200 mg	0.0008 M
Potassium Chloride (mw: 75 g/mol)	400 mg	0.005 M
NaHCO3 (mw: 84.01 g/mol)	2.2 g	0.026 M
NaCl (mw: 58.44 g/mol)	6.8 g	0.117 M
NaH2PO4H2O (mw: 138 g/mol)	140 mg	0.001 M
D-Glucose (Dextrose) (mw: 180.156 g/mol)	1 g	0.006 M
Phenol Red (mw: 354.38 g/mol)	10 mg	0 M

EBSS (magnesium, calcium, phenol red) (pH 7.0) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 200 mg of CaCl₂ to the solution.
3. Add 200 mg of MgSO₄-7H₂O to the solution.
4. Add 400 mg of Potassium Chloride to the solution.
5. Add 2.2 g of NaHCO₃ to the solution.
6. Add 6.8 g of NaCl to the solution.
7. Add 140 mg of NaH₂PO₄H₂O to the solution.
8. Add 1 g of D-Glucose (Dextrose) to the solution.
9. Add 10 mg of Phenol Red to the solution.
10. Adjust solution to final desired pH using HCl or NaOH
11. Add distilled water until volume is 1 L.

Glycine (0.1 M, pH 2.2) Preparation

Glycine is a bulking agent that can help promote protein stability. To prepare 1L of Glycine (0.1 M, pH 2.2) the following components are required;

Component	Amount	Concentration
Glycine (Sigma G7126) (mw: 75.07 g/mol)	7.5 g	0.0999 M

Glycine (0.1 M, pH 2.2) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 7.5 g of Glycine (Sigma G7126) to the solution.
3. Adjust the pH to 2.2 with HCl.
4. Add distilled water until volume is 1 L.
5. Autoclave.

Glycine-HCl Buffer (0.1 M, pH 3.0) Preparation

This low-pH buffer is used extensively with affinity chromatography, particularly with proteins and antibodies. It is also used for cell culture and other biological applications. To prepare 1L of Glycine-HCl Buffer (0.1 M, pH 3.0) the following components are required;

Component	Amount	Concentration
Glycine (mw: 75.07 g/mol)	7.5 g	0.1 M
Hydrochloric acid (mw: 36.46 g/mol)	832 mg	0.02 M

Glycine-HCl Buffer (0.1 M, pH 3.0) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 7.5 g of Glycine to the solution.
3. Add 832 mg of Hydrochloric acid to the solution.
4. Adjust solution to final desired pH using HCl or NaOH
5. Add distilled water until volume is 1 L.

Glycine-Sodium Hydroxide Buffer (0.08 M, pH 10) Preparation

A common buffer with good buffering capacity, often used in cellular biology. This buffer will slightly inhibit alkaline phosphatase enzymatic activity due to the configuration of the glycine amino group at higher pH levels. Due to this inhibition, it is recommended to use horseradish peroxidase (HRP) conjugates with this buffer.

To prepare 1L of Glycine-Sodium Hydroxide Buffer (0.08 M, pH 10) the following components are required;

Component	Amount	Concentration
Glycine (mw: 75.07 g/mol)	6.01 g	0.08 M
NaOH (mw: 40 g/mol)	2.05 g	0.0513 M

Glycine-Sodium Hydroxide Buffer (0.08 M, pH 10) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 6.01 g of Glycine to the solution.
3. Add 2.05 g of NaOH to the solution.
4. Adjust solution to final desired pH using HCl or NaOH
5. Add distilled water until volume is 1 L.

MOPSO Buffer (0.5 M, 6.9 pH) Preparation

MOPSO buffer is one of the Good’s zwitterionic buffer with an approximate pH range of 6.5-7.9. It has a good buffering capacity but does react with iron ions. MOPSO is used for a variety of assays that require physiological pH.

To prepare 1L of MOPSO Buffer (0.5 M, 6.9 pH) the following components are required;

Component	Amount	Concentration
MOPSO, Free acid (mw: 225.26 g/mol)	112.63 g	0.5 M

MOPSO Buffer (0.5 M, 6.9 pH) Preparation

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 112.63 g of MOPSO, Free acid to the solution.
3. Adjust solution to desired pH by 10N NaOH.
4. Add dH2O until volume is 1 L.

MOPS Buffer (10X) (0.2 M, pH 7) Preparation 

MOPS buffer is often used in polyacrylamide gel electrophoresis. To prepare 1L of MOPS Buffer (10X) (0.2 M, pH 7) the following components are required;

Component	Amount	Concentration
MOPS free acid (mw: 209.26 g/mol)	41.86 g	0.2 M
Sodium Acetate (mw: 82.03 g/mol)	4.1 g	0.05 M
Na2EDTA (mw: 372.24 g/mol)	3.72 g	0.01 M

MOPS Buffer (10X) (0.2 M, pH 7) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 41.86 g of MOPS free acid to the solution.
3. Add 4.1 g of Sodium Acetate to the solution.
4. Add 3.72 g of Na₂EDTA to the solution.
5. Adjust solution to desired pH using NaOH (typical pH = 7)
6. Add dH2O until volume is 1 L.

MES (0.5 M, pH 6) Preparation

MES buffer is a Good’s buffer that is remarkably stable both chemically and enzymatically. It has minimal UV absorbance and is often used as a running buffer for bis-tris gels. To prepare 1L of MES (0.5 M, pH 6) the following components are required;

Component	Amount	Concentration
MES free acid (mw: 195.24 g/mol)	97.62 g	0.5 M

MES (0.5 M, pH 6) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 97.62 g of MES free acid to the solution.
3. Starting pH for 0.5M MES solution is 3.23 For 1 L of 0.5 M MES, 13.6 ml of 10N NaOH is needed to adjust the pH to 6.0
4. Add dH2O until volume is 1 L.

Maleic Acid (1 M, pH 7.5) Preparation

Maleic acid buffer is commonly used for Southern blot preparation and as a washing buffer in numerous applications involving nucleic acids. It has a shelf life of 6 months to a year.

To prepare 1L of Maleic Acid (1 M, pH 7.5) the following components are required;

Component	Amount	Concentration
Maleic Acid (mw: 116.1 g/mol)	11.6 g	1 M
NaCl (mw: 58.4 g/mol)	8.8 g	5 M

Maleic Acid (1 M, pH 7.5) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 11.6 g of Maleic Acid to the solution.
3. Add 8.8 g of NaCl to the solution.
4. Add dH2O until volume is 1 L.
5. Sterilize by autoclaving at 15 psi for 20 minutes or by filtration. Store at room temperature. For washing applications, add 0.2% Tween-20 if desired.

Imidazole-HCl Buffer (0.05 M, pH 7.0) Preparation

Imidazole is a mild buffer that is useful for IHC applications in multiple research areas such as biochemistry and molecular biology. It has a good shelf-life and is well-tolerated by multiple cell types. To prepare 1L of Imidazole-HCl Buffer (0.05 M, pH 7.0) the following components are required;

Component	Amount	Concentration
Imidazole (mw: 68.08 g/mol)	3.404 g	0.05 M
HCl (mw: 36.46 g/mol)	886 mg	0.0243 M

Imidazole-HCl Buffer (0.05 M, pH 7.0) Preparation

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 3.404 g of Imidazole to the solution.
3. Add 886 mg of HCl to the solution.
4. Adjust solution to desired pH using HCl (typically pH ≈ 7.0).
5. Add distilled water until volume is 1 L.

Hydrochloric Acid-Potassium Chloride Buffer (0.1 M, pH 2.0) Preparation

This common buffer is used in multiple biology research fields, often for enzymatic or histochemical usage. It should ideally be prepared shortly before use, as it does not store well. To prepare 1L of Hydrochloric Acid-Potassium Chloride Buffer the following components are required;

Component	Amount	Concentration
KCl (mw: 74.55 g/mol)	7.45 g	0.1 M
HCl (mw: 36.46 g/mol)	772 mg	0.02 M

Hydrochloric Acid-Potassium Chloride Buffer (0.1 M, pH 2.0) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 7.45 g of KCl to the solution.
3. Add 772 mg of HCl to the solution.
4. Adjust solution to final desired pH using HCl or NaOH
5. Add distilled water until volume is 1 L.

HEPPSO (1 M, pH 7.85) Preparation

HEPPSO is a Good’s buffer used with protein separation in biological research. It is a Zwitterionic compound and binds strongly with copper ions. To prepare 1L of HEPPSO (1 M, pH 7.85) the following components are required;

Component	Amount	Concentration
HEPPSO (mw: 286.35 g/mol)	268.33 g	1 M

HEPPSO (1 M, pH 7.85) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 268.33 g of HEPPSO to the solution.
3. Adjust solution to desired pH with 10N NaOH.
4. Add dH2O until volume is 1 L.

PIPES Buffer (1 M, 6.8 pH) Preparation

PIPES buffer is one of the Good’s zwitterionic buffer with a pH range of 6.1-7.5. To prepare 1L of PIPES Buffer (1 M, 6.8 pH) the following components are required;

Component	Amount	Concentration
PIPES (mw: 302.37 g/mol)	302.37 g	1 M

PIPES Buffer (1 M, 6.8 pH) Preparation

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 302.37 g of PIPES to the solution.
3. Solubility of PIPES free acid is low in acidic range. Adjust solution to desired pH by 10N NaOH.
4. Add dH2O until volume is 1 L.

Phosphate Buffer (pH 5.8 to 7.4) Preparation

A simple phosphate buffer is used ubiquitously in biological experiments, as it can be adapted to a variety of pH levels, including isotonic. This wide range is due to phosphoric acid having 3 dissociation constants, (known in chemistry as a triprotic acid) allowing for formulation of buffers near each of the pH levels of 2.15, 6.86, or 12.32. Phosphate buffer is highly water soluble and has a high buffering capacity, but will inhibit enzymatic activity and precipitates in ethanol. The buffer is one of the most popular currently used, and is commonly employed in molecular and cell biology, chemistry, and material science, among many others.

To prepare 1L of Phosphate Buffer (pH 5.8 to 7.4) the following components are required;

Component	Amount	Concentration
Na2HPO4•7H2O (mw: 268.07 g/mol)	20.214 g	0.0754 M
NaH2PO4•H2O (mw: 137.99 g/mol)	3.394 g	0.0246 M

Phosphate Buffer (pH 5.8 to 7.4) Preparation Steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 20.214 g of Na₂HPO₄•7H₂O to the solution.
3. Add 3.394 g of NaH₂PO₄•H₂O to the solution.
4. Adjust solution to final desired pH using HCl or NaOH
5. Add distilled water until volume is 1 L.

MOPSO Buffer (0.5 M, 6.9 pH) Preparation 

MOPSO buffer is one of the Good’s zwitterionic buffer with an approximate pH range of 6.5-7.9. It has a good buffering capacity but does react with iron ions. MOPSO is used for a variety of assays that require physiological pH. To prepare 1L of MOPSO Buffer (0.5 M, 6.9 pH) the following components are required;

Component	Amount	Concentration
MOPSO, Free acid (mw: 225.26 g/mol)	112.63 g	0.5 M

MOPSO Buffer (0.5 M, 6.9 pH) Preparation 

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 112.63 g of MOPSO, Free acid to the solution.
3. Adjust solution to desired pH by 10N NaOH.
4. Add dH2O until volume is 1 L.

MOPS Buffer (10X) (0.2 M, pH 7) Preparation

MOPS buffer is often used in polyacrylamide gel electrophoresis. To prepare 1L of MOPS Buffer (10X) (0.2 M, pH 7) the following components are required;

Component	Amount	Concentration
MOPS free acid (mw: 209.26 g/mol)	41.86 g	0.2 M
Sodium Acetate (mw: 82.03 g/mol)	4.1 g	0.05 M
Na2EDTA (mw: 372.24 g/mol)	3.72 g	0.01 M

MOPS Buffer (10X) (0.2 M, pH 7) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 41.86 g of MOPS free acid to the solution.
3. Add 4.1 g of Sodium Acetate to the solution.
4. Add 3.72 g of Na₂EDTA to the solution.
5. Adjust solution to desired pH using NaOH (typical pH = 7)
6. Add dH2O until volume is 1 L.

MES (0.5 M, pH 6) Preparation 

MES buffer is a Good’s buffer that is remarkably stable both chemically and enzymatically. It has minimal UV absorbance and is often used as a running buffer for bis-tris gels. To prepare 1L of MES (0.5 M, pH 6) the following components are required;

Component	Amount	Concentration
MES free acid (mw: 195.24 g/mol)	97.62 g	0.5 M

MES (0.5 M, pH 6) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 97.62 g of MES free acid to the solution.
3. Starting pH for 0.5M MES solution is 3.23 For 1 L of 0.5 M MES, 13.6 ml of 10N NaOH is needed to adjust the pH to 6.0
4. Add dH2O until volume is 1 L.

Maleic Acid (1 M, pH 7.5) Preparation

Maleic acid buffer is commonly used for Southern blot preparation and as a washing buffer in numerous applications involving nucleic acids. It has a shelf life of 6 months to a year.

To prepare 1L of Maleic Acid (1 M, pH 7.5) the following components are required;

Component	Amount	Concentration
Maleic Acid (mw: 116.1 g/mol)	11.6 g	1 M
NaCl (mw: 58.4 g/mol)	8.8 g	5 M

Maleic Acid (1 M, pH 7.5) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 11.6 g of Maleic Acid to the solution.
3. Add 8.8 g of NaCl to the solution.
4. Add dH2O until volume is 1 L.
5. Sterilize by autoclaving at 15 psi for 20 minutes or by filtration. Store at room temperature. For washing applications, add 0.2% Tween-20 if desired.

Imidazole-HCl Buffer (0.05 M, pH 7.0) Preparation

Imidazole is a mild buffer that is useful for IHC applications in multiple research areas such as biochemistry and molecular biology. It has a good shelf-life and is well-tolerated by multiple cell types.

To prepare 1L of Imidazole-HCl Buffer (0.05 M, pH 7.0) the following components are required;

Component	Amount	Concentration
Imidazole (mw: 68.08 g/mol)	3.404 g	0.05 M
HCl (mw: 36.46 g/mol)	886 mg	0.0243 M

Imidazole-HCl Buffer (0.05 M, pH 7.0) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 3.404 g of Imidazole to the solution.
3. Add 886 mg of HCl to the solution.
4. Adjust solution to desired pH using HCl (typically pH ≈ 7.0).
5. Add distilled water until volume is 1 L.

Hydrochloric Acid-Potassium Chloride Buffer (0.1 M, pH 2.0) Preparation

This common buffer is used in multiple biology research fields, often for enzymatic or histochemical usage. It should ideally be prepared shortly before use, as it does not store well. To prepare 1L of Hydrochloric Acid-Potassium Chloride Bufferthe following components are required;

Component	Amount	Concentration
KCl (mw: 74.55 g/mol)	7.45 g	0.1 M
HCl (mw: 36.46 g/mol)	772 mg	0.02 M

Hydrochloric Acid-Potassium Chloride Buffer (0.1 M, pH 2.0) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 7.45 g of KCl to the solution.
3. Add 772 mg of HCl to the solution.
4. Adjust solution to final desired pH using HCl or NaOH
5. Add distilled water until volume is 1 L.

Potassium Phosphate (pH 5.8 to 8.0) Preparation

Potassium phosphate buffers, sometimes called Gomori buffers, consist of a mixture of monobasic dihydrogen phosphate and dibasic monohydrogen phosphate. These buffers have excellent buffering capacity and are highly soluble in water. However, potassium phosphate buffer reacts with some metal ions including calcium and magnesium, and inhibits enzymatic reactions. Additionally, all phosphates precipitate in ethanol, which can be deleterious to some nucleic acid preparations that typically use ethanol solutions. Potassium phosphate buffer is used in research fields ranging from molecular biology to botany, due to its adaptability and affordability.

To prepare 1L of Potassium Phosphate (pH 5.8 to 8.0) the following components are required;

Component	Amount	Concentration
K2HPO4 (mw: 174.18 g/mol)	16.282 g	0.0935 M
KH2PO4 (mw: 136.086 g/mol)	888 mg	0.0065 M

Potassium Phosphate (pH 5.8 to 8.0) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 16.282 g of K₂HPO₄ to the solution.
3. Add 0.888 g of KH₂PO₄ to the solution.
4. Add dH2O until volume is 1 L.

Trizma® Buffer (pH 7.0 to 9.2) Preparation 

Trizma® is a proprietary chemical buffer used similarly to Tris buffer. It is commonly used in protein extraction for many types of IHC assays as well as blot applications. It is used in sandwich ELISA protocols for protection of the antibodies, and in electrophoresis of nucleic acids.

To prepare 1L of Trizma® Buffer (pH 7.0 to 9.2) the following components are required;

Component	Amount	Concentration
Trizma HCl (mw: 157.6 g/mol)	1.011 g	0.0064 M
Trizma Base (mw: 121.14 g/mol)	11.337 g	0.0936 M

Trizma® Buffer (pH 7.0 to 9.2) Preparation 

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 1.011 g of Trizma HCl to the solution.
3. Add 11.337 g of Trizma Base to the solution.
4. Add distilled water until volume is 1 L.

Tris-citrate Buffer (pH 8.0) Preparation

Tris-citrate buffer can be used for electrophoresis of bcterial enzymes. To prepare 1L of Tris-citrate Buffer (pH 8.0) the following components are required;

Component	Amount	Concentration
Citric acid (mw: 192.19 g/mol)	16 g	0.0833 M
Tris (mw: 121.14 g/mol)	40.2 g	0.3318 M

Tris-citrate Buffer (pH 8.0) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 16 g of Citric acid to the solution.
3. Add 40.2 g of Tris to the solution.
4. Add distilled water until volume is 1 L.

Tris-Buffered Saline (TBS, 0.1 M) Preparation

Tris-Buffered Saline (TBS) is commonly used for washing in many applications, such as cell culture, IHC, WB, and ELISA. To prepare 1L of Tris-Buffered Saline (TBS, 0.1 M) the following components are required;

Component	Amount	Concentration
NaCl (mw: 58.44 g/mol)	8 g	0.1368 M
KCl (mw: 74.55 g/mol)	200 mg	0.0027 M
Tris (mw: 121.14 g/mol)	12.1 g	0.0999 M

Tris-Buffered Saline (TBS, 0.1 M) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 8 g of NaCl to the solution.
3. Add 200 mg of KCl to the solution.
4. Add 12.1 g of Tris to the solution.
5. Adjust the pH to 7.4 with HCl.
6. Add distilled water until volume is 1 L.

Tris Buffer (1 M, pH 7.2) Preparation 

Tricine is derived from the amino acids tris and glycine. It is a dipolar ion (Zwitterionic) and hydroxyl radical scavenger, and is used extensively for SDS-PAGE applications for small proteins. Tricine buffer is also commonly used for electrophoresis procedures.

To prepare 1L of Tris Buffer (1 M, pH 7.2) the following components are required;

Component	Amount	Concentration
Tris base (mw: 121.14 g/mol)	121.14 g	1 M

Tris Buffer (1 M, pH 7.2) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 121.14 g of Tris base to the solution.
3. Adjust solution to desired pH using HCl (typically pH ≈ 7.0).
4. Add distilled water until volume is 1 L.

Tricine (1 M, pH 8.05) Preparation

Tricine is derived from the amino acids tris and glycine. It is a dipolar ion (Zwitterionic) and hydroxyl radical scavenger, and is used extensively for SDS-PAGE applications for small proteins. Tricine buffer is also commonly used for electrophoresis procedures.

To prepare 1L of Tricine (1 M, pH 8.05) the following components are required;

Component	Amount	Concentration
Tricine (mw: 179.17 g/mol)	179.17 g	1 M

Tricine (1 M, pH 8.05) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 179.17 g of Tricine to the solution.
3. Adjust solution to desired pH with 10N NaOH.
4. Add dH2O until volume is 1 L.

TES Buffer (1 M, 7.5 pH) Preparation

TES buffer is one of the Good’s zwitterionic buffer with a pH range of 6.8-8.2. To prepare 1L of TES Buffer (1 M, 7.5 pH)  the following components are required;

Component	Amount	Concentration
TES (mw: 229.25 g/mol)	229.25 g	1 M

TES Buffer (1 M, 7.5 pH) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 229.25 g of TES to the solution.
3. Adjust solution to desired pH by 10N NaOH.
4. Add dH2O until volume is 1 L.

TBS (1 M, pH 7.4) Preparation

Tris-Buffered Saline (TBS) is a popular isotonic buffer used for multiple applications, including as a washing buffer in immunoassays of all kinds. It is one of the default buffers for antibody preparation. For other antibody preparation materials, click here.

To prepare 1L of TBS (1 M, pH 7.4) the following components are required;

Component	Amount	Concentration
NaCl (mw: 58.4 g/mol)	8 g	0.137 M
KCl (mw: 74.5 g/mol)	200 mg	0.0027 M
Tris base (mw: 121.14 g/mol)	3 g	0.0248 M

TBS (1 M, pH 7.4) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 8 g of NaCl to the solution.
3. Add 200 mg of KCl to the solution.
4. Add 3 g of Tris base to the solution.
5. Adjust solution to desired pH with HCl (typically 7.4).
6. Add dH2O until volume is 1 L.

TB Buffer Preparation

To prepare 1L of TB Buffer the following components are required;

Component	Amount	Concentration
EDTA (mw: 292.24 g/mol)	370 mg	0.001 M
KCl (mw: 74.55 g/mol)	13.64 g	0.183 M
NaCl (mw: 58.44 g/mol)	2.74 g	0.047 M
Phenylmethylsulfonyl fluoride (PMSF) (mw: 174.19 g/mol)	170 mg	0.001 M
Tris-HCl (mw: 157.6 g/mol)	1.21 g	0.01 M

TB Buffer Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 370 mg of EDTA to the solution.
3. Add 13.64 g of KCl to the solution.
4. Add 2.74 g of NaCl to the solution.
5. Add 170 mg of Phenylmethylsulfonyl fluoride (PMSF) to the solution.
6. Add 1.21 g of Tris-HCl to the solution.
7. Adjust pH to 6.8.
8. Add distilled water until volume is 1 L.
9. Sterilize by filtration.

TAE (1 M, pH 8.6) Preparation

TAE buffer is commonly used with nucleic acids for agarose electrophoresis applications. For the associated fluorescent probes for gel electrophoresis, click here.

To prepare 1L of TAE (1 M, pH 8.6) the following components are required;

Component	Amount	Concentration
Tris base (mw: 121.14 g/mol)	242 g	2 M
Disodium EDTA (mw: 336.21 g/mol)	18.61 g	1 M
Acetic Acid (mw: 60.05 g/mol)	59.955 g	1 M

TAE (1 M, pH 8.6) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 242 g of Tris base to the solution.
3. Add 18.61 g of Disodium EDTA to the solution.
4. Add 59.955 g of Acetic Acid to the solution.
5. The 1x TAE solution is 40mM Tris, 20mM Acetate and 1mM EDTA and typically has a pH around 8.6 (do not adjust).
6. Add dH2O until volume is 1 L.

SSC buffer (20X) (3 M, pH 7) Preparation

Saline-sodium citrate (SSC) buffer is often used as a hybridization buffer for DNA and RNA, particularly for Northern and Southern blots.

To prepare 1L of SSC buffer (20X) (3 M, pH 7) the following components are required;

Component	Amount	Concentration
NaCl (mw: 58.4 g/mol)	175.3 g	3 M
Sodium Citrate (mw: 258 g/mol)	77.4 g	0.3 M

SSC buffer (20X) (3 M, pH 7) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 175.3 g of NaCl to the solution.
3. Add 77.4 g of Sodium Citrate to the solution.
4. Adjust solution to desired pH with 14 N HCl (typically 7.0).
5. Add dH2O until volume is 1 L.

Sodium Phosphate Solution Preparation

To prepare 1L of Sodium Phosphate Solution the following components are required;

Component	Amount	Concentration
Na2HPO4 (mw: 141.96 g/mol)	70 g	0.493 M
NaH2PO4 · H2O (mw: 137.99 g/mol)	30 g	0.2174 M

Sodium Phosphate Solution Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 70 g of Na2HPO4 to the solution.
3. Add 30 g of NaH2PO4 · H2O to the solution.
4. Add distilled water until volume is 1 L.
5. Autoclave and store at room temperature.

Sodium Borate Buffer (1 M, pH 8.5) Preparation

Sodium Borate Buffer is a buffer that is used to maintain the pH within a relatively narrow range. It is isotonic and has a strong bactericidal effect, and can be used to dilute substances or in coating procedures. To prepare 1L of Sodium Borate Buffer (1 M, pH 8.5) the following components are required;

Component	Amount	Concentration
Boric Acid (H3BO3) (mw: 61.83 g/mol)	61.83 g	1 M
NaOH (mw: 39.997 g/mol)	10 g	0.25 M

Sodium Borate Buffer (1 M, pH 8.5) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 61.83 g of Boric Acid (H3BO3) to the solution.
3. Add 10 g of NaOH to the solution.
4. Add dH2O until volume is 1 L.

Sodium Acetate (3 M, pH 5.2) Preparation 

Sodium Acetate (3 M) is commonly used during DNA and RNA extraction. Sodium acetate, which is a salt, helps precipitate nucleic acids. To prepare 1L of Sodium Acetate (3 M, pH 5.2) the following components are required;

Component	Amount	Concentration
sodium acetate (mw: 82.03 g/mol)	246.1 g	3 M

Sodium Acetate (3 M, pH 5.2) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 246.1 g of sodium acetate to the solution.
3. Adjust the pH to 5.2 with glacial acetic acid. Allow the solution to cool overnight. Adjust the pH once more to 5.2 with glacial acetic acid.
4. Add distilled water until volume is 1 L.
5. Filter-sterilize the solution.

Potassium Phosphate Buffer (1 M, pH 6.5) Preparation

Potassium phosphate buffers, sometimes called Gomori buffers, consist of a mixture of monobasic dihydrogen phosphate and dibasic monohydrogen phosphate. These buffers have excellent buffering capacity and are highly soluble in water. However, potassium phosphate buffer reacts with some metal ions including calcium and magnesium, and inhibits enzymatic reactions. Additionally, all phosphates precipitate in ethanol, which can be deleterious to some nucleic acid preparations that typically use ethanol solutions. Potassium phosphate buffer is used in research fields ranging from molecular biology to botany, due to its adaptability and affordability.

To prepare 1L of Potassium Phosphate Buffer (1 M, pH 6.5) the following components are required;

Component	Amount	Concentration
KH2PO4 (mw: 136.09 g/mol)	95 g	0.698 M
K2HPO4 (mw: 174.18 g/mol)	52.5 g	0.3014 M

Potassium Phosphate Buffer (1 M, pH 6.5) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 95 g of KH2PO4 to the solution.
3. Add 52.5 g of K2HPO4 to the solution.
4. Adjust the pH to 6.5.
5. Add distilled water until volume is 1 L.
6. Filter sterilize and store at room temperature.