Hello everyone,
I am trying to evaluate by western blotting proteins secreted by cells to the culture medium (in cell cultures). I found a precipitation protocol with TCA/Acetone (https://www.its.caltech.edu/~bjorker/Protocols/TCA_ppt_protocol.pdf) but I was wondering if I should I first separate the floating cells in the culture medium to have just the soluble protein in the supernatant or should I add directly TCA solution to the medium taken from the cell culture?
I hope to read your answers and thanks in advance :)
https://www.researchgate.net/post/What-is-the-best-way-to-isolate-and-purify-a-protein-from-a-conditioned-medium
https://www.researchgate.net/post/Protein-purification-from-culture-supernatant
https://www.researchgate.net/post/How-to-concentrate-the-total-proteins-in-cell-culture-medium-secreted-by-the-cells
The above RG discussions may be related to your question. There are also many similar discussions that may be of your help in RG or you could use another search.
Hello Diego Benitez Riquelme
You should first collect the conditioned media containing the floating cells, dead cells and cellular debris and centrifuge this media at 900× g for 10 min at 4 °C so that the soluble proteins remain in the supernatant and other unwanted components (cells) are pelleted down. Then proceed further.
Best.
Yes, you should spin all of the debris out first. But you can also avoid precipitation and simply concentrate your media. This concentrated media can then be run on a gel for western blot
Diana Baxter I have 2 questions
1) how do you concentrate the media?
2) I was thinking if I concentrate the media at the end I will have not only the secreted proteins but also the components of the medium (salts, aminoacids, glucose, vitamins, etc.). Could these components affect the results? or maybe the final blot could be a little bit "dirty"?
Thank you :)
You can use Pierce protein concentrators. You can choose what volume you want to concentrate (eg. 1ml, 6ml, 15ml) and also what molecular weight cut off you want to use. I have previously used 5ml media concentrated down to 400ul and get more protein than when extracting from cells. I have not done many western blots using these samples, but have never had a problem with media components interfering when looking at proteins such as collagens.
@Diego, If you centrifuge your medium to remove debris and concentrate it as described by others, above, you can then run two gels or gel lanes (2D vs 1D), one for virgin medium (never saw cells) and the other for depleted medium (containing cell secretions and reduced by consumption original ingredients), you should be able to see the new spots (possible secretions) and possibly the amount of decrease in media proteins (e.g. serum proteins).
Thank you very much for your answers Shin Murakami , Diana Baxter and Christopher Lange :) I will follow recommendations as much as I can and hpe to get good results !
With TCA precipitation protocol, when should I measure the protein concentration? Because here (https://www.its.caltech.edu/~bjorker/Protocols/TCA_ppt_protocol.pdf) is only the precipitation method and at the end adding Sample Buffer for SDS PAGE.
TCA precipitation may denature your proteins. I think it's better to try acetone precipitation.
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