Does anyone know an accurate technique to quantify intracellular protein concentration besides ELISA? I need a reliable and highly quantitative method to measure protein concentration to gather data from cell culture experiments and incorporate them into my model of protein-protein interactions.
Are you talking total protein concentration? If so you can use the BCA method from Pierce.
No, I am talking about a specific protein, I guess it has to be something involving antibodies to make it specific for a particular protein.
If it is an enzyme then you can get the concentration by measuring activity. If it does not have any enzymatic acivity, specific spectral properties or specific ligand binding, then ELISA is probably the best way to go.
To quantify a particular intracellular protein in monocytes i use flow cytometry. I stain whole blood for monocytic markers (CD45,CD14, CD16 etc), then permeabilse and fix the cells to carry out intracellular staining. after acquisition through flow cytometry i can gate the monocyte population and analyse the expression of the protein via the mean fluorescence intensity (MFI).
We used this method in this study:
Article Expression of the Kynurenine Pathway in Human Peripheral Blo...
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