The modified figure and legend were reprinted by permission from Springer Nature: Copyright 2019 (Color figure online) A protein with dual topology would display an increase in the amount of labeled protein after disruption in the upper panel and reduced amount of labeling detected after disruption in the lower panel. The target protein is immunoprecipitated and resolved by sodium dodecyl sulfate-polyacrylamine gel electrophoresis and biotinylated protein is detected using avidin-horse radish peroxidase with the predicted and observed results shown on the right. Labeling by MPB that cannot be blocked by such AMS treatment is an independent verification of a residue that is facing the interior. Labeling by MPB that can be blocked completely by pretreatment with AMS is an independent verification of an outside facing residue (bottom panel). Both halves are either kept intact (–) or disrupted (+) by sonication or detergent treatment to expose the interior cysteine followed by treatment with MPB to specifically label the previously inaccessible internal cysteine residue. Half of the sample is treated with the detectable thiol reagent 3-(N-maleimidylpropionyl) biocytin (MPB) to specifically label the externally exposed cysteine (top panel), and the other half is treated with the non-detectable thiol reagent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) (bottom panel) to protect external cysteines during subsequent MPB lableing. A target membrane protein containing a single cysteine replacement exposed either to the exterior (blue) or interior (red) side of a cell membrane, membrane vesicle or proteoliposome is shown. The same mechanism may be important in transfections with other polyplexes that require high charge ratios for transfection.General strategy for SCAMTM using impermeable thiol reagents. The intracellular elimination of pDNA was faster in the presence of GAGs and, despite improved transfection, free PEI reduced pDNA association with the cells.įree PEI is essential for minimizing the undesirable binding of polyplexes to cell-surface GAGs that have a negative impact on transfection. PEI polyplexes with free carrier mediated transfection in both normal and GAG-deficient cells because free PEI overcomes the inhibitory effect of cell-surface GAGs on transfection. This indicates that the cell-surface GAGs inhibit transfection by purified polyplexes. In the absence of cell-surface GAGs, polyplexes without free PEI had high transfection efficacy. Transfection activity of polyplexes without free PEI in cells expressing cell-surface GAGs was low even though pDNA was delivered to cells. The total amount of cell-associated plasmid DNA (pDNA) delivered by polyplexes was determined by quantitative real-time PCR and transgene expression was determined using β-galactosidase and luciferase assays. Although positively charged PEI polyplexes are known to interact with anionic cell-surface glycosaminoglycans (GAGs), the exact role of free PEI in such interactions is unclear.Ĭhinese hamster ovary wild-type cells and mutants lacking cell-surface GAGs were transfected with marker genes using PEI polyplexes with and without free PEI. The excess of PEI gives polyplexes a positive surface charge that plays a role in polyplex binding on the cell membrane. Polyethylenimine (PEI) polyplexes mediate efficient gene transfer only at high +/- charge ratios at which free noncomplexed PEI is present.
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