Protein Extraction from cell culture media

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Tamsinash
Tamsinash's picture
Protein Extraction from cell culture media

 I am attempting to quanitfy a certain protein released by microglia into the DMEM cell culture media after their stimulation and activation. The protein was not significant enough to show up in my Western. How do you concentrate the protein levels enough to quantify them? What is the protocol for this? Thank you in advance. 

Ruderus
Ruderus's picture
 Hi there,

 Hi there,

there are several ways to concentrate proteins when you have a large volume of liquid. 

You can use simple concentrators, they are smal columns that allow you to reduce the liquid volume and thus concentrate your samples. The AMICON ULTRA are sold by Millipore, but there are many other to chose from.

Alternatively, you can perform a TCA PRECIPITATION (Tri-cloro-acetic acid). It is a little more time consuming but is relatively simple and you can find protocols online. 

Whatever you choose to do, remember that the cell culture medium contains a lot of proteins (from the serum) and they are going to be A LOT MORE  than the proteins you are interested to look at... so my suggestion is to try to have you protein secreted in a serum free medium and then concentrate it with a column.

Good luck!
 

g a
g a's picture
 Hi Tamsinash

 Hi Tamsinash

I would suggest that you first lyophillize the medium,  and then follow it with concentration using centrifugal filters or TCA - Acetone precipitation. Lyuphillization will enable you to concentrate the large amount of medium while using the concentrators  you will be only able to do less volume.  

Prefer using Serum free medium. That way you are sure that all the proteins are from one source.

Tamsinash
Tamsinash's picture
 Hi, 

 Hi, 
Thanks for the input! 
I have actually found an article that fetal calf serum in DMEM has  been shown to affect certain protein regulators in the microglia. So in addition to your suggestions to have serum-free medium, I am looking to remove it. How would one go about this? Do you culture the cells in serum media and then switch to serum-free when plating? Wont the cells be starved for nutrients and that could affect results no? 
Thanks again!

Chin Fen Teo
Chin Fen Teo's picture
Hi Tamsinash,

Hi Tamsinash,

I think you do need to try different conditions and see how your cells look. You should not remove the serum during the routine culturing/maintaining/plating steps, but do get rid of the serum while setting up your experiments.

Whether the cell will be deprived of nutrients/growth hormone is very cell type specific. Some cells will survive (and go into G0 stage) in the absence of serum for 24 to 48 hours before they start dying, some cells will immediately undergo apoptosis in the absence of serum (starting from 8 to 16 hours). So you really have to setup a few conditions and see how long microglia can survive in the absence of serum.

If your cells are totally unhappy in the absence of serum (ie. undergo apoptosis even before your protein of interested is being release into the culture medium), you can try to switch them in Opti-MEMI (from Invitrogen/Gibco, cat# 31985)- it is a defined formula that can support cell growth under reduced serum condition.

Good luck.

Ruderus
Ruderus's picture
 Hi Tamsinash,

 Hi Tamsinash,

I agree with Pippuri, you need to run some tests to determine what your cells can stand and what they cannot.

I would grow my cells in medium with serum, and then change to serum free medium just before you do your stimulation. I don t know how long stimulation and secretion require, but if you can wrap it up in a few hours (4-6 ), your cells should not suffer too much... just make sure they are well fed and happy when you remove  the serum. 

Another option for you would be to immuno-isolate your secreted protein from the medium using a specific antibody, possibly in a column format assay, so that you can pass the medium on the column several times.  You should also save the flow-through and quantify how much of you protein is still there. This will require some tests to set the best condition.