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Figure 1 | Proteome Science

Figure 1

From: Antibody cross-linking and target elution protocols used for immunoprecipitation significantly modulate signal-to noise ratio in downstream 2D-PAGE analysis

Figure 1

Type of cross-linker affects specificity and yield of immunoprecipitated protein. Dynabeads Protein A were bound to anti-UNG PU59 antibodies (Ab) and either incubated directly with HeLa cell extract, or cross-linked with DMP, BS3 (BS), or half the recommended amount of BS3 (BS1/2) prior to incubation with HeLa cell extract for 4 hours. Subsequent to washing, bound proteins were eluted in SDS-PAGE loading buffer and analyzed by: A) Silver stained gel; B) Western blot. The bright region in lane 2 of the western blot is caused by excessive binding of HRP-conjugated antibody to the primary antibody eluting from the non-crosslinked beads, causing exhaustion of the luminol substrate.

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Proteome Science

ISSN: 1477-5956