Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins.

Abstract

Immunofluorescence microscopy of cultured animal cells is often performed after detergent permeabilization of formaldehyde-fixed cellular membranes so that antibodies may have access to intracellular antigens. A comparison was made of the ability of several detergents, after formaldehyde fixation, to affect localization of intracellular proteins or to permeabilize different organelles to antibodies. Saponin, a detergent-like molecule that can permeabilize cholesterol-containing membranes, was also used. Four monoclonal antibodies were found to have a bright, discrete fluorescence localization with saponin alone, but were almost undetectable when the cells were treated with nonionic detergents such as Triton X-l00 or NP-40. These immunoglobulin G antibodies included two against lysosomal membrane glycoproteins, one against an integral GOLDENTHAL, HEDMAN, CHEN, AUGUST, WILLINGHAM Materials and Methods Cell cultures. Four cell lines were examined: fibroblastic mouse NIH 3T3 and Swiss 3T3 cells, baby hamster kidney (BHK) cells, and ' + 3, discrete and very bright;

Toward a Confocal Subcellular Atlas of the Human Proteome

“…It is noteworthy that even though the antibody concentration was normalized there were a higher number of unspecific and negative stainings for low stock concentration compared with higher concentration suggesting that high concentrations of specific antibodies after immunization is an indicator of good antibody quality. However, it is worth pointing out that unspecificity of the staining can also be associated with the fixation/permeabilization protocol used as shown previously (11,12).…”

“…1. Although saponin yielded excellent images for proteins localized in the cytoplasm, our results suggest that stronger detergents, such as Triton X-100, are needed to stain proteins localized to mitochondria or nuclei (data not shown) (11). The Triton permeabilization was therefore chosen as the standard procedure for the high throughput effort.…”

“…There are many different options available, all with different advantages and disadvantages (11,12). Several different alternatives to suit a high throughput approach were investigated, 2 and examples of PFA/Triton X-100 and PFA/saponin are shown in Supplemental Fig.…”

Profiling Subcellular Protein Phosphatase Responses to Coxsackievirus B3 Infection of Cardiomyocytes

“…Detergents rapidly extract proteins but lyse subcellular compartments indiscriminately (59). Cell lysis itself can inactivate protein phosphatases by oxidation, and phosphatase activity in lysates may be offset by constitutive kinase activities that co-extract (60,61).…”

“…Adherent cells are gently permeabilized without mechanical disruption by using a saponin extraction buffer to permeabilize cells by displacing cholesterol selectively in cell membranes (62). The nuclear envelope has negligible cholesterol and thus only small nuclear proteins (Ͻ 40 kDa) that freely diffuse through nuclear pore complexes will be released with saponin extraction (59,63). Cells are washed with saponin-containing PBS and then incubated briefly with phosphate-free Nonidet P-40 (NP40) extraction buffer to solubilize lipid bilayers, including the nuclear envelope.…”