I'm collecting supernatant as samples and measuring certain proteins released by cells/tissues after treatment. I would appreciate suggestions on how to standardize supernatant in running Westerns and ELISA. I am aware that running these assays using LYSATES we use total protein content measured by BCA assay or other protein assays. But what about if you are using supernatants where we don't expect intracellular proteins to be present? What should be used as a measure of uniformity in loading the wells?
I was precipitating proteins with organic solvents such as acetone, TCA..After precipitation I use to centrifuge and concentrate the protein in pellet.
I understand your suggestion. By doing so, are you suggesting that the proteins are being released in the supernatant and are coming from the tissue/cells?
I am interested in your protocol. can you please expound or provide more details on how to go about it.
Dear Ryan,
Cells/tissues may secrete a lot of cytokine/growth factors into the culture medium.If you are looking at a specific protein then you can buy ELISA kits specific to the protein and species.If you are looking to find out what proteins these cells/tissues secrete,you can then look to go for a protein array(eg.RayBio array).
Hope this helps!
I'm not trying to profile all the proteins released in the cytokine. I am interested in measuring inflammatory cytokines. I am aware of ELISA kits which could be used to do that. However, i also want to run westerns on the supernatant which is a more affordable protocol to do than opting an elisa. My problem is, since i am not using cell/tissue lysates, i won't be able to use Actin or GAPDH as housekeeping proteins to normalize the bands of my target proteins because these are structural proteins which are obtained usually when cells/tissues are lysed. Is there any alternative housekeeping that i can use to normalize band intensity? Can I use tissue weight in normalizing the band intensity? Is it ok to NOT have a housekeeping protein in reporting data of westerns? These are my specific concerns.
Dear Ryan,
This is an interesting question. One way to do this is that you have to standardize a consistently secreted cytokine in the cells you are working with within donors/lots of cells. This consistently secreted cytokine could be your internal control. This warrants extreme caution, in my experience secretory cytokine secretion varies a lot within donors or batches.The simpler way out is an ELISA.It is probably necessary only to have an appropriate molecular weight ladder.
Refer to this paper...this was but then published way back in 1991...
http://www.ncbi.nlm.nih.gov/pubmed/1902865
What about using the same volume of the supernatant or if possible calculate density/amount of cells present?
Tomas, can normalization be done by simply having uniform volume of supernatant from tissue explants? We dont do this in running westerns of lysate as we load based on protein content of the lysate to have uniform (normalized) loading per well, as normally done and reported in most papers. In which case we compute the volume of the samples that would come up with the desired amount of protein to load per well. Can I do the same for supernatant? What you suggests of loading uniform volume of supernatant could result in varying amounts of protein per well, isn't it?
Amount of protein per million cells could be a better way than considering per well volume of media. But then it is important to keep the media volume constant.
Well, if you expect your protein to be the main ingredient in supernatant and you expect changes in it, than you cannot normalize to proteins. It that case would be best IMHO normalization to volume or cells.But counting (measuring) the cells won't be easy I guess?
Hi,
Here the same question. I'm culturing primary cells (from patients) and we want to compare the amount of secreted factors by this cells. Is there a way to standardize culturing of primary cells or measuring the amount of secreted factors?
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