![]() Methods for protein normalization include use of internal housekeeping protein controls such as GAPDH, β-tubulin, β-actin, or cyclophilin B, exogenous loading controls, or total protein normalization. Protein normalization is used to correct for this variability and is a critical step in obtaining reliable and reproducible quantitative western blot data. In western blotting, variable results can be caused by unequal protein sample concentration, inconsistent sample loading onto the gel, and/or transfer variation during electroblotting. No-Stain Labeling Buffer, 20X (2 x 20 mL each storage 15-30⁰C) No-Stain Derivatizer (800 µL storage -20⁰C) No-Stain Activator (800 µL storage -20⁰C) The No-Stain Protein Labeling Reagent forms covalent bonds with a portion of the lysine amino acid side chains on all protein samples Tunable sensitivity-Increasing incubation time or doubling the reagent concentration produces a proportionally stronger signal for improved detection of low abundance proteins.Ī standard kit will label 40 mini gel-sized membranes or 40 mini gels, or a combination of the twoĬompatible with any gel type (precast or pour your own) or gel chemistry (Bis-Tris, Tris-glycine, Tris-acetate, Tricine)Ĭompatible with all downstream immunodetection steps with chemiluminescence or fluorescence-based detectionĬompatible with a wide range of imagers that have UV or fluorescence light sources, for example, the iBright FL1500 Imaging System.Sensitive and stable signal-Protein bands are detected down to 20 ng and the signal is compatible with downstream antibody detection.Broad linear dynamic range-1–80 µg total protein loaded per well.Accurate total protein normalization-Imaging and total protein normalization analysis take only minutes with the iBright FL1500 Imaging System.Flexible visualization-Use a wide-range of excitation sources, including UV and green fluorescence light.The reaction time is 10 minutes for gels and transferred membranes alike. NEW Faster protocol-Mix, incubate and image.Compatible with all gel chemistries and with downstream gel staining, protein transfer, immunoblotting and mass spectrometry analysis. Versatile-Use for fast total protein labeling of gels and membranes.Additional data should be considered for a more accurate adjustment.Ġ.2~0.4 mg/ml of each band of protein is stored in a mixture of solution that contains: 20 mM Trisphosphate at pH 7.5, 2% SDS, 0.2 mM Dithiothreitol, 3.6 M Urea, and 15% (v/v) Glycerol.ĥμl of IRIS11 Prestained Protein Ladder (PMI11) resolves 11 bands in 4-20% SDS-PAGE (Tris-glycine buffer) and after Western blotting to PVDF membrane. ![]() Note: The molecular weight of each protein (kDa) was measured against an unstained protein ladder in every electrophoresis condition. 2.5 μl per well for general Western transferring. Refer to the IRIS11 Prestained Protein Ladder patterns in various electrophoresis conditions:ģ μl or 5 μl per loading for clear visualization during electrophoresis on 15-well or 10-well mini-gel, respectively. Guide for Molecular Weight Estimation (kDa) The IRIS11 Prestained Protein Ladder keeps track of the size and separation of proteins during SDS-polyacrylamide gel electroph oresis, approximating the target protein size and validating the Western transfer efficiency on PVDF, nylon, or nitrocellulose membranes. The 11 recombinant proteins are covalently coupled with blue chromophore, while 2 red bands at 70 kDa and 260 kDa, a green band at 15 kDa and a newly designed peacock green band at 60 kDa serve as reference bands. The IRIS11 Prestained Protein Ladder is a combination of 11 pre-stained proteins with molecular weights from 3 to 260 kDa. ![]()
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