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Fluorescent western blot with secondary antibodies conjugated to IRDye® is quantitative and provides wider dynamic range than enzyme-based approaches, making it best option for quantifying relative protein abundance.
For guidance on sample preparation, running gels, and transferring protein from gel to membrane, see our general western blot protocol.
Incubate membranes with 10 mL of the primary antibody solution on a rocking platform. Optimal dilution and incubation times should be determined using serial dilutions and a time course.
Place the wash trays and petri dishes on a rocker during the washes ensuring that the blots are washed separately.
Between washes, dilute the secondary antibodies in antibody dilution buffer according to the WB experimental plan (see Table 1 below). Ensure that the secondary antibody is thoroughly mixed with the dilution buffer.
All secondary antibodies are light sensitive and should be covered with aluminium foil or similar during incubation.
After incubation, discard the secondary solution and repeat washing procedure in Step 2, keeping the membrane in the original Petri dish or wash tray.
Primary raised in
Close the equipment lid. Scan strips accordingly to equipment instructions. Remove the membranes from the scan bed and clean the scan bed with 70% ethanol using a cotton lint-free cloth.