In my job, I transient transfected Active-wnt3a construct into Hek293T and Hek293 for expression , I collected three batch(fresh medium 5ml in 10cm dish for 24hr)
conditioned medium and concentrated in 500 µl ,but it didnt have any effect when it treatment 20µl for TopFlash repoter assay!!!!
Can any one tell me what can i do?
I presume you used serum free medium if you concentrated - this can reduce the synthesis and secretion of proteins from cells so you may have to incubate longer before harvest. Try also using complete medium without concentrating. Also, 5 mL medium in a 10 cm culture dish seems too low a volume to me - should be at least 10 mL. Do you have enough cells? Make sure your cell monolayer is confluent - does not matter that cells become overgrown or if the medium turns a bit acidic. YOu should repeat harvest up to 2 times by adding fresh medium. Did you check transfection activity? include a small amount of GFP plasmid for transfection and look at cells under fluorescent microscope next day. How much Wnt3a plasmid did you transfect?
Could it be that your secreted wnt-protein gets trapped by extracellular matrix components on the Hek cell surface (e.g. heparan sulfate proteoglycans)? Maybe you could try another cell line? ATCC is selling mouse fibroblasts (stable transfected) that they describe for generating conditioned media (CRL2647).
If you want to check for biological activity of your constructs you could try to transiently co-transfect TopFlash with your wnt-plasmid, incubate some time in serum-free medium, lyse the cells and assay the cell extract - this should (at least to some extent) give you increased basal luciferase activity of the co-transfected cells in comparison to only TopFlash transfected cells...?
Good luck,
Christian
Hii
Although I am not an expert, I have attached you a paper where they have used L cells to purify Wnt3a proteins, if nothing works you might want to try this original protocol.
good luck.
Ganesh
Wnt conditioned media is popular because recombinant Wnt-3a is such poor quality. There is now a reliable alternative available which is making conditioned media obsolete: Check our PODS-wnt3a
http://www.cellgs.com/products/podsand8482-human-wnt-3a.html
L-cells are better for making Wnt conditioned medium, although I don't know exactly the reason.
As reference, have a look here:
https://web.stanford.edu/~rnusse/assays/W3aPurif.htm
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