Formaldehyde sensitivity of a GFAP epitope, removed by extraction of the cytoskeleton with high salt.
We have used cytofluorometry to examine the formaldehyde sensitivity of the binding of a monoclonal antibody (MAB) to its epitope on glial fibrillary acidic protein in human malignant glioma cells in culture. When acetone-extracted whole cells or cytoskeletons, made by extracting with Triton in stabilizing buffer (Tsb), are fixed with formaldehyde, binding of the MAB Tp-GFAP1 to GFAP is abolished or greatly reduced. Fixation with the bifunctional protein crosslinking reagent dithiobis (succinimidyl propionate) (DTSP) has the same negative effect as formaldehyde. If cytoskeletons are further extracted with Tsb containing 250 mM ammonium sulfate (Thsb), fixation with formaldehyde or DTSP has reduced or no effect on the binding of Tp-GFAP1. The data are consistent with the hypothesis that aldehyde sensitivity of Tp-GFAP1 is caused by the crosslinking of a second protein to GFAP that blocks the binding of the MAB to its epitope. This putative blocking protein is part of the Triton-insoluble cytoskeleton, but it begins to be solubilized in 50 mM ammonium sulfate and it is largely removed in 250 mM ammonium sulfate (Thsb). SDS-PAGE shows that extraction with Thsb also removes a large number of proteins from the cytoskeleton, one of which could be the blocking protein. A second antibody to GFAP, designated Tp-GFAP3, was raised against cytoskeletons which had been fixed with DTSP and in which the epitope recognized by Tp-GFAP1 was presumably blocked. Tp-GFAP3 is not sensitive to fixation by either formaldehyde or DTSP.
D000911:Antibodies, Monoclonal; D003599:Cytoskeleton / Q000502:physiology*; D000939:Epitopes / Q000302:isolation & purification*; D005557:Formaldehyde / Q000494:pharmacology*; D005904:Glial Fibrillary Acidic Protein / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D008856:Microscopy, Fluorescence; D013388:Succinimides
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005904:Glial Fibrillary Acidic Protein; D013388:Succinimides; D005557:Formaldehyde; C011240:dithiobis(succinimidylpropionate)