Detection of Western blots using fluorescent labeled secondary antibodies can allow for more accurate quantification and two-color multiplexing using primary antibodies from different species. Phosphorylation and total protein levels may be determined on the same membrane, and regulation of different targets at different molecular weights can be examined simultaneously. There can also be significant long-term advantages in terms of digital data storage as well as the reduction of costs for chemiluminescent substrates, x-ray film, and darkroom facilities.
Western blot analysis of extracts from COS cells, untreated or treated with either U0126 (#9903) (10 µM for 1h) or TPA (#4174) (200 nM for 10m), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) Rabbit mAb (#4370) and p44/42 MAPK (Erk1/2) (3A7) Mouse mAb (#9107).
Western blot analysis of extracts from Raw264.7 cells, untreated or LPS-treated (1 µg/ml for 6 h), using iNOS Antibody (#2977).
Many CST antibodies are being tested by Western blot using near-infrared dye-conjugated secondary antibodies and a slightly modified immunoblotting protocol. We have found that omitting Tween-20 during the blocking step is the only significant change necessary. Full details can be found in our Fluorescent Western Immunoblotting Protocol (Primary Ab Incubation In BSA) and our Fluorescent Western Immunoblotting Protocol (Primary Ab Incubation In Milk). We have also found that prestained standards such as our Prestained Protein Marker, Broad Range (Premixed Format) (#7720) are autofluorescent at near-infrared wavelengths.
Two-color Western blots require primary antibodies from different species and appropriate secondary antibodies labeled with different dyes. If the primary antibodies require different primary antibody incubation buffers, test each primary individually in both buffers to determine the optimal one for the dual-labeling experiment. Some antibody pairs may not work together due to overlapping epitopes, interference caused by primary and/or secondary antibodies, or incompatibilities in primary antibody incubation buffers.