Invitrogen
anti-ISG54 (IFIT2) polyclonal antibody
PA3845
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The goal of this experiment is to determine the induction of ISG54 (known as IFIT2) in macrophages upon virus infection using IFA and western blotting methods. In the figure A, virus infected-bone marrow-derived macrophages were fixed at 12 h.p.i, permeabilized with 0.1% Triton X-100 and stained for ISG54 (green) and nucleus (blue). The data show the cytoplasmic location of ISG54. In the figure B, infected-cells were harvested at either 6 or 12 h.p.i, and total 20ug cell lysates were separated on 10% SDS-PAGE gel and probed with anti-ISG54 antibody. The results show this antibody has a high specificity. Non-specific band was not observed in western blot.
Western Blot
Mouse primary macrophages cell lysate 20ug
1:500 dilution for IFA; 1:2000 for WB. 4 degree overnight incubation
WB: 5% non-fat milk in TBST at RT for 1 hour; IFA: 2% BSA
WB: 1: 10000 dilution in TBST with goat anti mouse IgG at RT for 1 hour; IFA: donkey anti-rabbit IgG alexa fluor 488 (1:1000)
NA
Membranes were washed and visualized for chemiluminescence in Western Lightning Plus-ECL solution; IFA: cells were imaged with confocal microscope.
IFA: ISG54 was clearly stained in virus-infected or neighboring cells. Mock-infected cells show a basal level expression of ISG54 in macrophages.WB: There was only one band displaying on the membrane, indicating a high specificity. There was a significant induction of ISG54 in virus-infected cells than uninfected cells.
High specificity
Pricey
high specificity even it's a rabbit polyclonal Ab.