Immunofluorescent staining controls - (Sep/05/2006 )
Hi, I am trying to identify suitable control proteins for my immunostaining. I will be looking at localization of a protein, and so wanted to stain for an ER protein (BIP), a plasma membrane protein and a cytosolic protein. What are commonly used control proteins/antibodies for this purpose? Thanks!!
depends strongly of kind of cell type, for instance, for epithelial cells most try to distinguish between apical and basolateral membrane utilizing for each specific markers; there are also some compartment-specific low molecular weight markers;some useful stains are provided by Molecular probes now Invitrogen
I asume you are going to do immunofluorescence and confocal microscopy to assess co-localization of your control protein (that of which you certainly know the localization) and the protein(s) you are studying.
There is a bunch of proteins that you can use for subcellular compartment identification. What is best for you depends on the cell lineage and the cell physiological condition you are studying.
As a general rule you would use enzymes and/or receptors with known subcellular distribution under defined conditions (this also helps analyze protein translocation if any under different stimuli).
As an example , protein disulfide-isomerase can be used to label the intraluminal space of ER and glycosyltransferases for Golgi identification. Caveolins are integral plasma membrane proteins, associated with protein scaffolding to the plasma membrane, and can therefore be used to label plasma membranes in different cell types. Last, but not least, several glycolytic enzymes can be used to label the cytoplasmic compartmente. However, bear in mind that caveolin provide a scaffold to some glycolytic enzymes (e.g., PFK) that are localized to the plasma membrane. This means that some glycolytic enzymes may be targeted to the plasma membrane if the adequate stimuli are provided.
Hope this helps. Best regards,