Glycophorin having one highly hydrophobic domain is able to span a phospholipid bilayer membrane only
Answers
Glycophorin on human red cells is a useful model for phage selection, because of the ready access to both intact red cells, and the purified glycophorin. It has been possible to select with the phage displayed scFv on the red cells and confirm the binding activity on the purified glycophorin in ELISA wells. It could be adapted for other antigens and cell types with only minor modifications.
About 4 × 107 erythrocytes/ml in PBS bearing the desired antigen (in this case, glycophorin) are reacted with two volumes of a suspension of phage displaying Fab fragments of antibodies, and rocked for 30 min. About 10 volumes of ice-cold PBS are then added, and the erythrocytes pelleted by centrifugation and washed several times in PBS. The bound phage are eluted from the packed cells with 20 μl of elution buffer (pH 3.5), centrifuged at 6500 r.p.m. and the supernatant transferred to another Eppendorf tube and immediately neutralized.
It will sometimes be helpful to initially deplete a random library for phage that are reactive with erythrocyte membrane antigens with undesired specificities. This may sometimes be achieved if a source of cells can be found that lack the desired antigen, but are similar to the selecting cells in other respects. For example, erythrocytes may be selected from a donor who lacks glycophorin, and the phage library absorbed on these cells. This may be done by incubating about 1012 phage with the packed erythrocytes for 30 min at room temperature on a rocking platform, and then removing the cells by centrifugation. The supernatant, containing the depleted phage, may then be used for a round of positive selection on cells possessing the desired antigen. This procedure may help to reduce the amount of screening required to isolate rare clones.
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Glycophorin having one highly hydrophobic domain is able to span a “phospholipid bilayer membrane” only once.
Explanation:
Glycophorin, a noteworthy erythrocyte membrane protein, displays hydrophobic and hydrophilic association. Glycophorin contains a layer implanted with helix made out of hydrophobic (or uncharged) amino acids. The anticipated length of this α helix (3.75 nm) is only adequate to traverse the hydrocarbon center of a “phospholipid bilayer”.
The hydrophobic side chains structure Van der Waals communications with the greasy acyl chains and shield the polar carbonyl (C=O) and amino (NH) gatherings of the peptide security, which are all hydrogen-attached to each other.