Polymethanal (paraformaldehyde)

From OpenWetWare

Jump to: navigation, search



Recipes for polymethanal buffers

Some people make 16 or 24% stocks to be diluted at a later point. But the solid polymethanal / PFA is very hard to dissolve at these high concentrations. Stocks or working solutions can be preserved by freezing for later use. Otherwise, methanal reacts with itself to form methanol and methanoate (acidification).

PFA 4%

for 50ml

2.0 g PFA
+5 ml H2O
+drop of NaOH (0.5-1.0M)
+45 ml PBS
(correct pH to 7.4 with HCl)

PFA 1%

for 10ml

0.1 g paraformaldehyde powder in small glass tube
+0.5 ml distilled water
+drop of 0.5-1.0M sodium hydroxide

heat to ~80°C for 2-3 mins
shake in water bath until PFA dissolved (beaker of very hot water)

+9.5ml PBS
correct pH with HCl if necessary

Can also be solved by heating to 80°C for 3 hours.

Too many names


Here's a list of equivalent terms: polymethanal, polyoxymethylene, polyformaldehyde, paraformaldehyde, paraform, polytrioxane,.. (regards from Babel) Common abbreviations are PFA, pMeO.

CAS number = 30525-89-4

Comparison with methanal


Chemistry of fixation

The aldehyde group, especially of methanal, is very reactive. It readily combines with amino groups to amides or with alcohol groups to esters. These reactions are mostly irreversible. Unreacted molecules have to be removed prior to subsequent experiments since they easily damage proteins like antibodies.

Problems with Fixation

There are issues in fixing free GFP in the cytoplasm of cells. Firstable 4% PFA reduces GFP fluorescence to some extend, but it is tolerable for most experiments. 4% PFA can also destroy the subcellular localization of GFP, i.e. exclusion from the nucleus. In our hands the fixation with more than 2% PFA or in combination with 0.5%< glutaraldehyde or 0.5%< acrolein destroys inclusion/exclusion of IRF3-GFP from the nucleus. Moreover 2% PFA fixed cells expressing IRF3-GFP loose integrity after 2-5 hours post fixation during storage in PBS 1x, meaning that cytoplasm containing GFP leaks out of the cells and remains as a bubble outside the cell. It can be washed away but does not distribute in the PBS buffer on its own. This issue was not solved by higher/lower PFA concentrations during fixation, by storage in H2O/PBS1x/PBS2x/fixation solution/permeabilization of cells after fixation with Tx100 or Saponin 0,2%.

Polymethanal safety data sheets



Personal tools