I am trying to purify some secreted protein in culture medium containing 10% FBS. But I need to know what is the amount of protein in the medium to do this. I will be using the Bradford Assay for the quantitation. However, I am afraid that the FBS may make it difficult to quantitate. I would like to know if it will be a problem?
You can use media containing FBS that hasn't been put on cells and use it as your 'Blank'. When you do your calculations, start by subtracting your reading of your blank from your actual samples.
DEar Desmond Wong
i think that a methods that asses the total protein will be not accute since the media itself due to the presence of FBS will be already rich of proteins and your protein will represent only a small % of the total protein and will be confused in he high background.
YOu have 2 possibilities:
1) If you protein, is a recombinant protein which contain a tag for affinity purifications, you can perform a small scale purification of a fraction of the surnatant and quantify the protein after purification
2) If a specific monoclonal antibody able to bind the protein is commercially available you can try to quantify it by several approaches as:
- BLI
- ELISA
- WB
but to perform absolute quantfication with thos approaches you need to built a calibration line with a purified protein sample.
good luck
ciao
Manuele
Hello Desmond Wong
Yes, FBS will make it difficult to quantitate your protein of interest. The conditioned media will contain FBS-derived bovine protein, albumin being the most abundant protein present in FBS. The depletion of albumin is conventionally conducted by using antibodies. There is no way to remove high-abundance bovine proteins. It is difficult to narrow the dynamic range of protein concentrations in the conditioned supernatant by a simple pre-treatment.
Instead of using FBS, can you not switch over to serum free media (SFM) or chemically defined media consisting of only purified organic and inorganic components?
The use of SFM represents an important tool that allows cell culture to be done with a defined set of conditions as free as possible of confounding variables, and easier purification and downstream processing.
Best.
Even with multiple measurements and statistical procedures, FBS at this percentage will give you so much background noise that you won't be able to measure your protein of interest. You could try to remove BSA and Immunoglobulins by affinity purification, but I doubt that this will improve the quality of your data, and you will have to do a lot of QC to prove the effectiveness and accuracy of what you are doing. Depending on the size and properties of your protein, analytical HPLC could work, but method development will require a lot of effort. If you have access to 2D-DIGE and MS or comparable methods, they might be ideal (possibly in combination with the removal of BSA and Immunoglobulins).
For scalability, a sandwich ELISA based procedure might be the most reasonable way to go, you also may follow the whole purification procedure in this way.
If you have access to a good antibody pair, you best bet would be to perform a quantitative sandwich ELISA. Alternatively, you could perform BLI/SPR as a quantitative measure of specific protein. The high albumin content of the media will prevent the use of total protein comparisons.
Thank you everyone for the suggestions. Greatly appreciated. Unfortunately there is no antibody pair that I can use which makes ELISA difficult. Also there is no serum free medium that is good for the cells I am using for long term culture. If I just use serum free RPMI or DMEM the cells changes its phenotype which is not desirable.
Yes it has a His-tag. I guess I can do a sandwich ELISA with the His-tag antibody as one of the antibodies. Thank you.
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