IEF standards: use 5 µl per mini gel for Coomassie staining or 0.5 µl for silver staining to yield 50 or 500 applications, respectively. The protein standard is supplied in a ready-to-use format for direct loading onto gels no need to heat, reduce, or add sample buffer prior to use. * 2-D standards: use 2.5 µl per mini gel for Coomassie staining (200 applications) or 0.5–2.5 µl for silver staining (up to 1,000 applications) use 1.0–5.0 µl for full-length gels (16–20 cm) to yield 100 or up to 500 applications, respectively. SeeBlue Plus2 Pre-Stained Standard contains 10 proteins (4250 kDa): 8 blue-dyed and 2 with contrasting colors, for easy and quick evaluation of electrophoresis and western transfer efficiency. 2-D SDS-PAGE protein standards provide calibrated references for protein pI and molecular weight in the second dimension. IEF and 2D protein standards are a mixture of native proteins with isoelectric points (pI) ranging from 4.45 to 9.6, providing reproducible pI calibration in native PAGE or agarose IEF gels. Bio-Rad’s Stain-Free Western Blotting Workflow facilitates speed and validation at each step of a western blotting experiment from running gels to quantifying proteins. Rinse the blot with 15 ml TBST at RT for 5 min. Incubate the blot in the primary antibody and blocking buffer solution at 4C overnight with gentle agitation. Life Science Group General Protocol for Western Blotting Protein separation by gel electrophoresis 1. Dilute the primary antibody 1:1,000 in 10 ml blocking buffer. Not intended for human or animal diagnostic or therapeutic uses.IEF and 2D Electrophoresis Protein Standards Bulletin 6376 Ver C US/EG 17-0657 05 Web. The apparent molecular weight (kDa) of each protein has been determined by calibration against unstained protein standards supplemental data should be considered for more accurate adjustment in different electrophoresis conditions. The 2-D Doctor is a self-help guide developed by Bio-Rad that enables you to identify and troubleshoot your 2-D gel issues. Troubleshooting 2-D Electrophoresis Gels with 2-D Doctor. Under suggested conditions, Unveil Unstained Protein Ladder resolves 12 major bands in 4-12%Bis- Tris Gel(MES buffer) and after Western blotting to the nitrocellulose membrane. Our self-help troubleshooting guide covers solutions to many common and not-so-common western blotting issues and helps your blots look their best. All prestained protein ladders are compatible for use in western blotting. Western blotting protein standards can be used for both fluorescent visualization and colormetric or chemiluminescent immunodetection on western blots. 2.5 μl per well for general Western transferring.Ģ0 mM Tris-phosphate(pH 7.5 at 25☌), 2 % SDS, 0.2 mM Dithiothreitol, 3.6 M Urea, and 15 % (v/v) Glycerol. Protein Standards & Ladders Thermo Fisher Scientific - US. The ladder is supplied in the gel loading buffer and is ready to use.ģ μl or 5 μl per loading for clear visualization during electrophoresis on 15-well or 10-well mini-gel, respectively. The Unveil Unstained Protein Ladder is designed for monitoring protein separation during SDS-polyacrylamide gel electrophoresis and verifying Western transfer efficiency on membranes (PVDF, nylon, or nitrocellulose) and for approximating the size of proteins. The proteins resolve into clearly identifiable bands when separated on the SDS-PAGE gel (MES buffer), with the intensified 25kDa and 85kDa protein bands serving as the two reference bands. The Unveil Unstained Protein Ladder is a mixture of 12 unstained recombinant proteins covering a wide range of molecular weights from 10 to 200 kDa.
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