May 23, 2026
Salting Out Purification of β-Lactoglobulin from Whey Protein Isolate
This protocol is a draft, published without a DOI.
- 1Carnegie Mellon University
Protocol Citation: Andrew Ashmar 2026. Salting Out Purification of β-Lactoglobulin from Whey Protein Isolate. protocols.io https://dx.doi.org/
Manuscript citation:
Mailliart, P.; Ribadeau‐Dumas, B. Preparation of β‐Lactoglobulin and p‐Lactoglobulin‐Free Proteins from Whey Retentate by NaCI Salting Out at Low pH. Journal of Food Science 1988, 53 (3), 743–745. https://doi.org/10.1111/j.1365-2621.1988.tb08945.x.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working. High purity has been confirmed using HPLC.
Created: February 04, 2026
Last Modified: May 23, 2026
Protocol Integer ID: 242583
Keywords: lactoglobulin from whey protein isolate method, whey protein isolate method, lactoglobulin, whey protein, immunoglobulin, purification
Abstract
Method for isolating β-lactoglobulin from whey protein isolate by "salting out" of α-lactalbumin and immunoglobulins. Adapted from work done by P. Mailliart & B. Ribadeau-Dumans (1988).
Materials
Sodium ChlorideVWR Whey Protein Isolaten/a Hydrochloric AcidFisher Scientific Deionized WaterMerck Millipore
Protocol materials
Hydrochloric AcidFisher Scientific
Deionized WaterMerck Millipore
Sodium ChlorideVWR
Whey Protein Isolaten/a
In a 250 mL Erlenmeyer flask, add24 g Whey Protein Isolaten/a to 160 mL Deionized WaterMerck Millipore under agitation (15% w/v). Use a large stir bar and a high agitation speed so the protein is well-dispersed.
10m
Once most of the protein is dissolved, use Hydrochloric AcidFisher Scientific to adjust the solution to pH 2.0.
15m
Add 11.2 g Sodium ChlorideVWR (7% w/v) to the solution under agitation. Some precipitate will form.
5m
Allow the solution to sit for 20 minutes to allow the protein to fully precipitate.
20m
Split the solution into four 50 mL centrifuge tubes. Centrifuge the solution at 3220 RCF (maximum speed) for at least 10 minutes.
15m
Collect the supernatant.
5m
OPTIONAL: Wash the precipitate with 6% NaCl (w/v) at pH 2.0 to recover any entrapped β-lactoglobulin. Centrifuge and collect the supernatant again.
25m
Desalt the solution by dialyzing against 3L of deionized water (12-14 kDa MWCO membranes recommended), changing the water at least four times with at least 3 hours between water changes. Make sure to stir the dialysis bath.
Author's Note: I recommend using a 4 liter beaker for this step. When skipping the washing step, I cut four standard-size dialysis tubes to reach from the bottom of the beaker to the 3 L mark. That should give enough length to fit the solution and secure the ends of the tubes.
1d
Pour the dialyzed solution into 50 mL centrifuge tubes. Freeze in liquid nitrogen (preferred) or a -80°C freezer, then lyophilize the contents until visibly dry (48-72h).
2d
Protocol references
Mailliart, P.; Ribadeau‐Dumas, B. Preparation of β‐Lactoglobulin and p‐Lactoglobulin‐Free Proteins from Whey Retentate by NaCI Salting Out at Low pH. Journal of Food Science 1988, 53 (3), 743–745. https://doi.org/10.1111/j.1365-2621.1988.tb08945.x.