The protein solubility calculator above answers the practical question at the front of almost every protein purification: how many grams of solid ammonium sulfate go into this volume to reach that percentage saturation. It handles the harder version too, where you are already at one saturation level and want the increment to reach a higher one, and it works backwards from a mass you have already weighed to the saturation it produces.
Arb Digital publishes free calculators that reproduce the published reference values rather than approximating them. The figures here match the standard ammonium sulfate saturation table to the nearest hundredth of a gram, and the page explains why the relationship is curved rather than proportional, which is the part that catches people out when they plan a two-step cut.
What This Protein Solubility Calculator Does
Salting out is the oldest protein purification method still in routine use, and it works because protein solubility falls as ionic strength climbs past a certain point. Different proteins fall out at different salt concentrations, so stepping the saturation upwards in stages separates them into fractions. Ammonium sulfate is the salt of choice because it is extremely soluble, cheap, and gentle on most proteins.
This page computes the solid mass to add. It reports the grams per litre required, the saturation step, the mass for a second cut, and the combined total, so a two-stage fractionation can be planned in one pass. The bar breakdown shows the mass for the four saturation bands most often used in practice at whatever volume you entered.
Two boundaries with adjacent tools matter here. The isoelectric point calculator deals with the other route to reduced solubility: taking the pH to the point where net charge disappears. Salting out and isoelectric precipitation are different mechanisms with different consequences for the protein, and the pI page explains that side. The protein concentration calculator converts an absorbance reading into mg/mL, which is what you use to measure recovery across each cut, and the protein molecular weight calculator supplies the extinction coefficient it needs.
How to Use It
- Enter your solution volume in millilitres. This is the volume before salt is added, which is the basis the reference table uses.
- Set the starting saturation. Zero for a fresh lysate or extract; the previous target when you are stepping up.
- Set the target saturation. Common first cuts are 30 or 40 percent, and common second cuts are 60, 70 or 80.
- Read the mass in grams and add it slowly with stirring, not all at once.
- Plan the second cut using the third grid figure, so you know the total salt requirement before starting.
The Formula and How It Is Calculated
The mass of solid ammonium sulfate required per litre of starting solution to move from saturation S1 to saturation S2, both as percentages, is g/L = 533 (S2 − S1) / (100 − 0.3 S2) at 20 °C. The numerator is the saturation increment scaled by the amount of salt a fully saturated solution holds; the denominator corrects for the volume the dissolved salt already occupies, which is why the relationship curves upward at high saturation.
The expression reproduces the standard published table. From zero, the amounts per litre are 113 g to reach 20 percent, 176 g for 30, 242 g for 40, 314 g for 50, 390 g for 60, 472 g for 70, 561 g for 80 and 657 g for 90 — the same values, to the nearest gram, as the reference figures used in the LibreTexts activity on purification via ammonium sulfate saturation.
Worked through: 50 mL of extract taken from 0 to 40 percent needs 242.3 g/L × 0.050 L = 12.11 g. Taking that same material on from 40 to 70 percent needs 533 × 30 / (100 − 21) = 202.4 g/L, so 10.12 g, giving 22.23 g in total. Note that the second step is not the same size as the first even though both span 30 to 40 saturation points; the correction term in the denominator has grown.
The reverse calculation rearranges the same expression: S2 = (100 g + 533 S1) / (533 + 0.3 g), where g is the grams per litre added. Entering 242.3 g/L from a start of zero returns exactly 40 percent, which is the round trip check.
Why Ammonium Sulfate and Not Some Other Salt
The choice is not arbitrary. Salts differ enormously in how strongly they drive proteins out of solution, and the ranking is the Hofmeister series, first described in the 1880s and still the working framework. Among cations, ammonium sits at the top of the precipitating end, ahead of potassium, sodium and lithium. Among anions, sulfate is near the top with fluoride, well ahead of chloride, while iodide and thiocyanate sit at the opposite end and actively increase protein solubility.
Ammonium sulfate therefore combines the two strongest common ions in a single salt. It is also remarkably soluble, reaching well over four moles per litre, which means high ionic strength is achievable at all; sodium chloride simply cannot reach the concentrations required. It is inexpensive, its solubility varies little with temperature, and it does not denature most proteins — in fact it often stabilises them, which is why purified proteins are sometimes stored as an ammonium sulfate slurry. The LibreTexts module on salting out sets out the Hofmeister ordering in full.
The mechanism is competition for water. At high salt concentration the ions are heavily hydrated and pull water away from the protein surface, exposing hydrophobic patches that then associate with one another rather than with solvent. That is a fundamentally different process from isoelectric precipitation, which removes the electrostatic repulsion instead. It is also why salting out is usually reversible: nothing about the protein's structure has necessarily been damaged, and dialysing the salt away often restores activity fully.
What the Percentage Actually Means
Percent saturation is a fraction of the salt a solution can hold at equilibrium, not a percentage by mass or by volume. Forty percent saturation means the solution contains forty percent of the ammonium sulfate that would be present in a fully saturated solution at that temperature. This is why the numbers are not proportional to each other and why a table is needed at all.
The temperature dependence is the part most often glossed over. Saturation is defined at a stated temperature, and the widely circulated tables exist in both zero degree and twenty degree versions with different coefficients. Most protocols say to work on ice, which is sensible for protein stability, while quoting masses from whichever table the author had to hand. The discrepancy is a few percent, which is usually tolerable but is not zero, and it is worth stating which table a protocol used. This page uses the 20 °C values and says so.
Volume is the other quiet assumption. The masses are per litre of the starting solution, and dissolving several hundred grams of solid noticeably increases the final volume. That matters when you calculate concentrations afterwards, and it is why the second cut is computed from the original volume rather than from the new one.
Where the Calculation Ends and the Experiment Begins
This is the honest limit of the page. The arithmetic tells you how much salt to add. It cannot tell you what saturation your particular protein comes out at, because that depends on the protein's surface chemistry, on the pH, on the temperature, on the total protein concentration and on what else is in the extract. Published cut points for a named protein are starting points from someone else's preparation, not constants.
Finding the right cut is an experiment. The standard approach is a pilot fractionation in ten or twenty percent steps across the whole range, assaying each pellet and each supernatant for total protein and for the activity you care about, and then narrowing the window. Total protein concentration matters more than people expect: a dilute extract needs a higher saturation to precipitate the same protein, because precipitation is a concentration-dependent aggregation process.
The practical technique also affects the outcome. Solid salt should be added slowly, in small portions, with continuous gentle stirring, and each portion allowed to dissolve before the next, because a locally saturated region will pull down proteins indiscriminately. Foaming denatures protein at the air-water interface, so vigorous stirring is counterproductive. Equilibration for a period after the final addition before centrifuging is standard, since precipitation is not instantaneous.
What Happens After the Pellet
An ammonium sulfate pellet is carrying a great deal of salt, and almost nothing downstream tolerates that. Ion exchange chromatography will not bind a sample at high ionic strength, because the salt competes for the resin, so the sample must be desalted first by dialysis or a buffer exchange column. Assays are affected too: high salt interferes with several protein quantitation methods and shifts many enzyme rates.
The one technique that welcomes it is hydrophobic interaction chromatography, which requires high salt to promote binding and elutes with a descending salt gradient. Loading an ammonium sulfate cut directly onto a hydrophobic column is therefore a natural pairing and saves a desalting step.
Whatever comes next, measure recovery at each stage. The point of a fractionation is enrichment, and enrichment is only visible if you know both the total protein and the activity before and after. The protein concentration calculator covers the total protein side, the enzyme activity calculator covers the activity side, and the ionic strength calculator quantifies the salt load you are asking the next step to cope with.
Arb Digital publishes hundreds of free calculators across chemistry, maths, finance and marketing — no sign-up, no limits. If something you need is missing, tell us and we will look at building it.
Browse All Free Tools Suggest a ToolCommon Mistakes to Avoid
- Treating saturation as linear — going from 40 to 80 percent needs more salt than going from 0 to 40, because the correction term grows with the target.
- Ignoring which temperature table a protocol used — the zero degree and twenty degree tables differ, and a cut on ice is not the same as the same number at room temperature.
- Adding the salt all at once — local supersaturation drags down protein indiscriminately and destroys the selectivity that is the entire point.
- Loading a salted sample onto an ion exchange column — it will not bind until the salt has been removed by dialysis or buffer exchange.
- Assuming a published cut point transfers — the saturation at which a protein precipitates depends on pH, temperature and total protein concentration in your own extract.
Related Free Tools From Arb Digital
The isoelectric point calculator covers the charge-based route to precipitation, and the protein molecular weight calculator and protein concentration calculator handle mass and quantitation. Use the molarity calculator and solution dilution calculator for the buffers, and the ionic strength calculator for the salt environment your next step has to tolerate. The full free online tools hub lists everything else.
Frequently Asked Questions
Multiply your volume in litres by 533 times the saturation increment, divided by 100 minus 0.3 times the target percentage. From zero to 40 percent that is 242 grams per litre, so 50 mL needs 12.11 grams.
It is the fraction of the ammonium sulfate a solution can hold at equilibrium at that temperature, not a percentage by mass or volume. Forty percent saturation contains forty percent of the salt a fully saturated solution would hold.
Because dissolved salt occupies volume, so the solution you are adding to is progressively less able to accept more. The correction term in the denominator grows with the target, which is why the second half of a range always costs more salt than the first.
Ammonium and sulfate both sit at the strongly precipitating end of the Hofmeister series, and ammonium sulfate is soluble enough to reach the ionic strengths required. Sodium chloride cannot reach those concentrations and its ions are far weaker at salting out.
Usually not. High ammonium sulfate concentrations often stabilise proteins rather than denature them, which is why purified proteins are sometimes stored as a salt slurry. Removing the salt by dialysis commonly restores full activity.
Yes. Saturation is defined at a stated temperature, and separate tables exist for zero and twenty degrees. This page uses the twenty degree values, so a protocol worked on ice from a zero degree table will differ by a few percent.
That has to be determined experimentally. It depends on the protein's surface chemistry, the pH, the temperature and the total protein concentration in the extract. A pilot fractionation in ten or twenty percent steps is the standard way to find it.
Redissolve it in buffer and remove the salt by dialysis or a desalting column before any ion exchange step or most assays. The exception is hydrophobic interaction chromatography, which requires high salt and can take the cut directly.
This calculator is provided for education and general reference. It describes how an ammonium sulfate saturation is computed and is not laboratory, handling or safety guidance; follow the protocols and risk assessments issued by your own institution.
