![]() In addition, UV imaging of the transferred membrane is much less sensitive than gel imaging, due to high autofluorescence of the membrane. If your target has many tryptophan residues, Stain-Free may block antibody to target binding, making accurate quantitation problematic. One limitation of this method includes chemical modification of your sample that may affect target detection and accurate normalization. With UV light, you can then visualize the sample proteins either on the gel or on the membrane. UV light causes these compounds to irreversibly cross-link with tryptophan residues present in your sample. This method uses a commercial pre-cast gel that contains trihalo compounds. γH2AX, the phosphorylated form of H2AX, is generated in response to DNA double-strand breaks. H2AX phosphorylation was analyzed with multiplexed pan-specific and phospho-specific antibodies and fluorescent immunoblotting. Phosphorylation of ATM used as its own internal loading control. Empiria Studio software automatically normalizes your target modification against the total target, regardless of modification, for you.įigure 1. Modified signal is then normalized to the total level of target protein. A modification-specific antibody specific to that modification’s epitope detects just the modified form of the target protein. Normalize a specific modification of your target against all target protein regardless of modification.Ī single antibody detects an unmodified epitope on the target protein, accounting for the total amount of target protein present (sometimes referred to as a pan-specific antibody). The target protein is used as its own internal loading control.
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