Barber RD, Harmer DW, Coleman RA, Clark BJ.(2012) Uncovering Suitable Reference Proteins for Expression Studies in Human Adipose Tissue with Relevance to Obesity. Pérez-Pérez R, López JA, García-Santos E, Camafeita E, Gómez-Serrano M, Ortega-Delgado FJ, Ricart W, Fernández-Real JM, Peral B.(2012) Stability of reference proteins in human placenta: General protein stains are the benchmark. Lanoix D, St-Pierre J, Lacasse A-A, Viau M, Lafond J, Vaillancourt C.(2014) CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis. Cazzalini O, Sommatis S, Tillhon M, Dutto I, Bachi A, Rapp A, Nardo T, Scovassi AI, Necchi D, Cardoso MC, Stivala LA, Prosperi E. American Society for Biochemistry and Molecular Biology. Aldridge GM, Podrebarac DM, Greenough WT, Weiler IJ.Stain-Free gel detection with UV light has a 35-fold linear dynamic range (1–35 µg) compared to UV detection on the membrane with a linear range of only 7-fold (10–70 µg). Stain-Free technology has a different linear range for gel and membrane detection. Stain-Free is more sensitive compared to Ponceau visible staining, but is still not as sensitive as near-infrared stains due to membrane autofluorescence.įigure 3. Finally, many proteins do not contain tryptophan residues, and therefore will not be modified or detected. 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). Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.The target protein is used as its own internal loading control. patents that may apply, see bd.com/patents.Īlexa Fluor and Pacific Blue are trademarks of Life Technologies Corporation. Not for use in diagnostic or therapeutic procedures.īD flow cytometers are Class 1 Laser Products.įor U.S. Spectral signatures are dependent on the cytometer configuration and settings used, and, as a result, results on individual instruments may differ from what is depicted herein.įor Research Use Only. Data for immunofluorescence fluorochromes were collected using conjugated antibodies.ĢCollection of pre-loaded configurations includes the following flow cytometers: BD Accuri™, BD FACSAria™ Fusion, BD FACSAria™ III, BD FACSCalibur™, BD FACSCelesta™, BD FACSJazz™, BD FACSLyric™, BD FACSMelody™, BD FACSVerse™, BD FACSymphony™ A1, BD FACSymphony™ A3, BD FACSymphony™ A5, BD FACSymphony™ A5 SE, BD FACSymphony™ S6, BD® LSR II, BD LSRFortessa™, BD LSRFortessa™ X-20.ģFluorochrome spectral signatures were generated on the respective instrument platform using recommended set-up protocols. 1Fluorochrome excitation and emission data were generated on a spectrophotometer and spectrofluorometer, respectively.
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