pH and Protein Solubility


Any change in the pH of a protein's environment will cause observable changes in the solubility of the protein. Solubility changes, in turn, reflect changes in the three-dimensional structure of the protein. The effect of pH on protein solubility explains why most enzymes function well at an optimum pH and why their activity decreases substantially at pH values other than the optimum. This demonstration examines the effect of pH on the solubility and structure of casein, a milk protein.


  • Protein
  • Isoelectric point
  • Solubility
  • pH


Casein, 1 g*
Hydrochloric acid, HCl, 2 M, 10 mL*
Sodium hydroxide, NaOH, 0.1 M, 25 mL*
Sodium hydroxide, NaOH, 2 M, 10 mL*
Universal indicator, 2 mL (includes pH color chart)*
Water, distilled or deionized
Balance, centigram
Beaker or flask, 600-mL
Magnetic stirrer and stirring bar
Pipets, Beral-type, graduated, 2*
*Materials included in kit.

Safety Precautions

Hydrochloric acid solution is a corrosive liquid and is toxic by ingestion and inhalation. Sodium hydroxide solution is corrosive and is especially dangerous to the eyes. Universal indicator is a flammable liquid—keep away from flames and heat. Avoid contact of all chemicals with eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Please review current Safety Data Sheets for additional safety, handling and disposal information.


Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. The casein solution may be stored at basic pH for several months. Alternatively, the solution may be rinsed down the drain with excess water according to Flinn Suggested Disposal Method #26b.


  1. Add 25 mL of 0.1 M sodium hydroxide and a stirring bar to a 600-mL beaker or flask and place the beaker on a magnetic stirrer. Add 225 mL of distilled or deionized water and stir at moderate speed. Add 1 g of casein and stir to dissolve. (The solution will be slightly cloudy or translucent.)
  2. (Optional) Add 1–2 mL of universal indicator to observe pH changes, if desired. Consult the universal indicator color chart for pH values.
  3. With rapid stirring, add 2 M hydrochloric acid in 0.5-mL increments using a graduated, Beral-type pipet. (The solution will turn cloudy, but will clear up again as the hydrochloric acid is dispersed. After 1–2 mL of acid has been added, the cloudiness will reach a maximum—this is the isoelectric point. The pH at the isoelectric point is 4–5.)
  4. Once the isoelectric point has been reached, pause just long enough to record observations (15–20 seconds.) Continue adding 2 M hydrochloric acid in 0.5-mL increments with stirring until the solution is clear again. (The cloudiness will fade and the precipitate will redissolve after the addition of another 2–3 mL of acid, when the pH drops below the isoelectric point, pH ≤2.)
  5. Continue to stir the solution. Reverse the process by adding 2 M sodium hydroxide in 0.5-mL increments using a clean, graduated, Beral-type pipet. (After 2–3 mL of sodium hydroxide has been added, the protein will precipitate out again at the isoelectric point.)
  6. Continue adding sodium hydroxide in 0.5-mL increments with stirring until the solution is clear again. (The solution will clear up after an additional 1–2 mL of sodium hydroxide has been added and the pH >10–12.)
  7. The process may be repeated using the same casein solution. This will give time to explain the observations.

Student Worksheet PDF


Teacher Tips

  • This kit contains enough materials to perform the demonstration as written at least seven times: 7 g of casein, of 100 mL of 2 M hydrochloric acid, 250 mL of 0.1 M sodium hydroxide, 70 mL of 2 M sodium hydroxide and 20 mL of universal indicator solution.

Correlation to Next Generation Science Standards (NGSS)

Science & Engineering Practices

Analyzing and interpreting data
Planning and carrying out investigations
Constructing explanations and designing solutions

Disciplinary Core Ideas

MS-PS1.A: Structure and Properties of Matter
MS-PS1.B: Chemical Reactions
HS-PS1.A: Structure and Properties of Matter
HS-PS1.B: Chemical Reactions
HS-PS2.B: Types of Interactions
HS-LS1.A: Structure and Function
HS-LS1.C: Organization for Matter and Energy Flow in Organisms

Crosscutting Concepts

Energy and matter
Structure and function

Performance Expectations

MS-PS2-2: Plan an investigation to provide evidence that the change in an object’s motion depends on the sum of the forces on the object and the mass of the object
MS-PS3-2: Develop a model to describe that when the arrangement of objects interacting at a distance changes, different amounts of potential energy are stored in the system.
HS-PS2-1: Analyze data to support the claim that Newton’s second law of motion describes the mathematical relationship among the net force on a macroscopic object, its mass, and its acceleration.
HS-PS2-4: Use mathematical representations of Newton’s Law of Gravitation and Coulomb’s Law to describe and predict the gravitational and electrostatic forces between objects.
HS-PS3-1: Create a computational model to calculate the change in the energy of one component in a system when the change in energy of the other component(s) and energy flows in and out of the system are known.
HS-PS3-2: Develop and use models to illustrate that energy at the macroscopic scale can be accounted for as a combination of energy associated with the motion of particles (objects) and energy associated with the relative position of particles (objects).

Sample Data


Answers to Questions

  1. Casein has both acidic side chains and basic side chains. At a high (basic) pH, ionization occurs in the acidic chains. At a very low (acidic) pH, protonation occurs in the basic chains. Do you think casein is most soluble with a net charge that is positive, negative or around zero? Why?

Casein is most soluble when it has either a highly positive or a highly negative charge. Bases ionize its acidic chains, resulting in a negative charge and acids protonate its basic chains, resulting in a positive charge. During the demonstration, there was the least amount of solid in the solution at the pH extremes. Therefore, casein is the least soluble when the net charge is zero.

  1. A protein’s isolectric point is the pH at which the protein has a net charge of zero. Approximate the isolectric point of casein.

The isoelectric point of casein is probably around 45, because that is when the solution was the cloudiest during the demonstration.


Casein is the principal protein in milk (80% of the total protein content). Casein is a phosphoprotein—it contains a large number of phosphate groups attached to the amino acid side chains in its polypeptide structure. The negatively charged phosphate groups are balanced by positively charged calcium ions and are responsible for the high nutritional calcium content in milk. Casein is almost completely insoluble in water at neutral pH (pH = 7).

Casein, like other proteins, is an ionic species containing amino groups and carboxyl groups on its terminal amino acids. It also contains a variety of other acidic and basic groups on the side chains of its non-terminal amino acids. The effect of pH on the solubility of casein reflects the ionization of the acidic and basic groups in its structure.

At high pH, casein will have a net negative charge due to ionization of all the acidic side chains (—CO2) in its structure. Because casein is ionized at high pH values, it is soluble in dilute sodium hydroxide solution.


At low pH, casein will have a net positive charge due to protonation of all basic side chains (—NH3+) in its structure. Because casein is ionized at low pH values, casein is also soluble in strongly acidic solutions.


At intermediate pH values, casein will contain roughly equal numbers of positively and negatively charged groups and the protein will have a net charge of zero. Casein is insoluble in neutral solutions because it is not charged under these conditions.


The solubility of a protein is usually at a minimum at its isoelectric point. The isoelectric point is defined as the pH at which a protein has a net charge of zero. For casein, due to the attached phosphate groups, the isoelectric point is close to pH = 4.


This activity was adapted from Flinn ChemTopic™ Labs, Volume 20, Biochemistry; Cesa, I., Editor; Flinn Scientific: Batavia, IL (2002).

Next Generation Science Standards and NGSS are registered trademarks of Achieve. Neither Achieve nor the lead states and partners that developed the Next Generation Science Standards were involved in the production of this product, and do not endorse it.