I am trying to separate membrane and cytosolic proteins from mouse hippocampus. Attached is the brief procedure. My question is how do I ensure that membrane fraction is devoid of cytosolic proteins and cytosolic fraction is devoid of membrane proteins? One marker can be alpha tubulin. It should be absent in membrane fraction. Indeed I found that to be the case. How about cytosolic fraction. Which protein should I look for as a marker of purity? (I understand that 100% separation is not posible, still I am looking for markers for quality control).
For personal experience in performing cell fractionation in cell coltures actin was better than tubulin (this seems more sticky to membranes) as marker of cytosol, while calnexin, BAP31 or derlin1 are perfect for membrane fractions. Soluble proteins contained in luminal compartments are often easily released during fractionation experiments and thus are not always good marker for membrane fraction.
In case of cytosolic protein you can use GAPDH, which is widely used in literature. However, in my view you should be careful about ER contamination in your membrane fraction (Na+K+ATPase is a good marker for membrane protein) and use appropriate maker for ER (e.g., Calnexin). You can also use Nuclear histone and MnSOD-Mitochondrial marker to further check the enrichment of your fractions.
Thank you Gianluca ans Sohail. Right now, I have N cadherin and E cadherin antibodies with me. Can I use these to check the presence of membrane proteins in my cytosolic fraction?
Actually, I am using the same buffer for dissolving both protein compartments i.e. pellet and supernatant (please have a look at the attached protocol). The buffer has 3 non ionic surfactants it it. I suspect that these surfactants specially triton (1%) and NP-40 are causing membrane proteins to leach out into supernatant fraction. What do you think about this?
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