I'm following a protocol to bind fluorescent ligand to receptors expressed on cells in a microplate. The protocol includes fixing cells with 4% PFA for 20 mins. This prevents cell loss during washing. But this ligand will only bind if the receptors are coupled to intracellular G proteins. Does fixing the cells kill these intracellular proteins?
Dear Prof. William K. Lim
No. Formaldehyde fixation is a widely used fixation method that results in low levels of shrinkage and good preservation of cellular structure for a wide range of cells and tissues and does not appear to result in significant structural changes to proteins.
When one fix cells with 4% PFA one preserves a snapshot of biomolecular transactions in vivo which is a widely used strategy in numerous techniques in biology and medicine. Due to its small size and high reactivity with a wide range of biological entities, PFA creates covalent cross-linking between biomolecules which includes proteins and nucleic acids. PFA non-selectively fixes or cross-links molecules in proximity to enable characterization of biomolecular interactions formed in living cells.
Regards,
Malcolm Nobre
Fixing cells while not destroying the majority of most protein epitopes, allowing the use of antibodies to probe fixed cells, it does largely 'kill' the enzymatic activity of most proteins.
Thanks for the responses. Dr Nobre I'm studying G protein coupled receptors, These will only bind an agonist ligand intracellular (membrane attached) G proteins move and couple with the receptors. Upon fixing, do you think these intracellular G proteins still move towards and physically to bind the receptors? Thanks.
Dear Professor,
The answer is "no". Cellular motion is immediately stopped by chemical fixation with PFA. The goal of fixation is to halt your cells decomposition and freeze cellular proteins and subcellular structures in place.
Is it possible for you to treat the cells with the ligand first and then fix the cells with 4% PFA. Detection may be carried out after fixation.
If you are worried about cell loss during the washing steps, make use of Ca2+ and Mg2+ containing PBS or use Poly-L-lysine coating of the surface. Also, you may work with buffer which is at 37°C. Avoid using cold buffer, for instance, during washing, as it may cause cell detachment.
Best wishes.
Thanks Dr Penketh. If that is the case, perhaps Dr Nobre's suggestion will resolve the problem- to fix cells only after ligand has bound. Provided the fixing will not lead to removal of bound ligand.
Dear Willian K. Lim,
Malcolm Nobre's suggestion should solve your problem.
Phil
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