Protocol for the Purification of Wnt Proteins

April 21, 2003, Written by Karl Willert

Additions on storage of tissue culture medium and purified protein, Sept 10, 2003

See also: Willert, K., Brown, J. D., Danenberg, E., Duncan, A. W., Weissman, I. L., Reya, T., Yates, J. R., and Nusse, R. (2003). Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448-452. Download a PDF file

Some modifications introduced by Kishida et al, (2004)

Note: The Wnt3A protein is being sold by R&D systems.

Necessary reagents
beta-catenin stabilization assay                                                                                                                                                                                            
Cells producing Wnt3A
Conditioned Medium
Blue Sepharose Fractionation
Heparin Cation Exchange
Purification in detail
The old text on Wnt production, references to earlier work

Necessary reagents:

Small scale: HiTrap Blue, 1ml and 5ml (Amersham Biosciences)

Large scale: column with a bed volume of 100 to 120ml Blue Sepharose HP (Amersham Biosciences, cat# 90-1000-22)

HiLoad 16/60 or 26/60 Superdex 200 prep grade (Amersham Biosciences)

HiTrap Heparin, 1ml (Amersham Biosciences)



Description of b-catenin stabilization assay:
Mouse L cells seeded in 96 well plates.
Add Wnt protein (dilution of conditioned medium or dilution of purified protein in complete medium).
Incubate cells 2 hours in 37C/CO2 incubator.
Aspirate medium.
Wash cells once with 1X PBS.
Aspirate PBS.
Lyse cells by adding 30 ul lysis buffer (1% Triton X-100, 150mM NaCl, 50mM Tris-HCl, pH8).
Add lysate to Laemmli buffer and boil 5 minutes.
Resolve 20ul of each sample by SDS-10% PAGE.
Transfer proteins to nitrocellulose and blot with mouse anti-b-catenin antibody.





Starting Material: Four day Wnt-3A conditioned medium from L-Wnt-3A; this medium contains about 100-200 ng Wnt-3A/ml. Filter, add detergent (Triton X-100 or CHAPS) to 1% final concentration, and re-filter just before applying the material to Blue Sepharose.


All purification steps are carried out at 4C. Do not freeze any fractions as this may be detrimental to the protein.


Small scale purification: 10-100 ml Wnt-3A CM on 1 ml or 5 ml HiTrap Blue Sepharose column. For the 5 ml HiTrap Blue column the binding capacity is about 100 ml CM for Wnt-3A.


Large scale purification: >100 ml Wnt-3A CM on 5ml HiTrap Blue columns in series or using a home packed column with Blue Sepharose HP (APB cat# 90-1000-22). For very large volumes (up to 4L) we use a sample pump for sample injections and a column packed to a bed volume of about 120ml Blue Sepharose HP. The Sepharose FF does not perform as efficiently as Sepharose HP.


1. Blue Sepharose

Small scale:

1-5ml HiTrap Blue column with up to 50ml Wnt CM + 1% Triton X-100 or CHAPS.

Eluent A: 1% CHAPS, 150mM KCl, 20mM Tris-HCl, pH7.5

Eluent B: 1% CHAPS, 1.5M KCl, 20mM Tris-HCl, pH7.5

Flow rate:1-2ml/min

Equilibrate column in Eluent A.

Inject Sample using Superloop.

Wash out unbound sample with 5 column volumes.

Elute bound sample with step gradient to 100% eluent B for 10 column volumes.

Collect 1-5ml elution fractions.


Large Scale

100-120 ml Blue Sepharose column with up to 4L Wnt CM + 1% Triton X-100 or CHAPS.

Eluent A: 1% CHAPS, 150mM KCl, 20mM Tris-HCl, pH7.5

Eluent B: 1% CHAPS, 1.5M KCl, 20mM Tris-HCl, pH7.5

Flow rate: 2-5ml/min

Equilibrate column in Eluent A.

Inject sample using sample pump.

Wash column with Eluent A with at least 4 column volumes.

Elute with Eluent B and collect 5-15 ml fractions.


Note: On a large Blue Sepharose column (CV ~120ml), Wnt3A elutes slowly despite the step elution, with several Wnt3A fractions trailing behind the main protein peak. The later fractions contain about half of the total Wnt3A protein but much less total protein. This material is much cleaner and will eventually produce the purest Wnt protein prep.


2. Gel Filtration

Sample: pooled Wnt containing fractions from Step 1 concentrated to 5 or 10 ml volume using Centricon or Amicon Ultra 30 ultrafiltration device (Amicon) depending on size of GF column.

Column: HiLoad 16/60 or 26/60 Superdex 200 prep grade

Eluent: 1X PBS, 1% CHAPS

Flow rate: 2ml/min

Inject Sample using Superloop.

Collect 5ml fractions for 16/60 and 10ml fractions for 26/60.

On HiLoad16/60 column Wnt3A will elute at 65-80 ml following a 5ml injection.

On HiLoad26/60 column Wnt3A will elute at 180-210 ml following a 10ml injection.


Note: The fraction numbers may change with varying buffer conditions and with performance/age of column. Especially note that lower concentrations of salt will cause the Wnt to be retained longer; at very low levels of salt (>50mM NaCl) the Wnt will emerge in the column volume fractions.


3. Heparin Cation Exchange Chromatography

Sample: pooled Wnt containing fractions from Step 2

Column: HiTrap Heparin, 1ml

Eluent A: 1X PBS + 1% CHAPS

Eluent B: Eluent A + 1 M NaCl.

Step elution from 0 to 100% Eluent B

Collect 1ml elution fractions.

We store the purified protein at 4 degrees C. It is then stable, although we sometimes add BSA to 100 micrograms per ml. We have also been able to freeze-dry the protein with retention of activity.

(what follows is the old and somewhat outdated text on this topic. It still contains some useful information)

There are numerous unpublished tales of failed attempts to produce secreted Wnt proteins in cell culture. In general, overexpression of the genes in cultured cells results in accumulation of misfolded protein in the ER [Kitajewski, 1992]. Secreted forms of Wnts can be found in the extracellular matrix or the cell surface [Bradley, 1990 ; Papkoff, 1990 ; Burrus, 1995], but efforts to solubilize this material have not been successful. Addition of suramin or heparin to cells can lead to a significant increase of Wnt protein in the medium [Bradley, 1990; Papkoff, 1990 , but this protein has not been shown to be biologically active [Burrus, 1995].

While under any circumstance most Wnt protein is cell bound, several systems have been developed that produce soluble forms.