Native PAGE

Several electophoretic methods (SDS-PAGE, native PAGE and lEF) showed that fraction Q2 was almost pure, some faint contaminating protein bands were found (Fig. 9). The major band upon SDS-PAGE was at 42 kDa. lEF indicated an isoelectric point at pH 4.3 for the most prominent band... 

SDS-PAGE was performed by the method of Laemmli [17]. The methods for native PAGE, isoelectric focussing, detection of esterase activity in electrophoresis gels, and assays for protein glycosylation have been described elsewhere [5]. 

SDS-PAGE analysis of purified FDH revealed two subunits and, in combination with native PAGE, suggested a a2 i structure. Selenium was not released from the protein during denaturing gel electrophoresis and was... 

Analyze 10 pi aliquot of the PCR product on a native PAGE (see Note 3). 

Anodic Discontinuous Poiyacryiamide Gei Eiectrophoresis (Native PAGE)... 

B 6. A protein mixture can be fractionated either by native PAGE or by denaturing, SDS-PAGE, before Western blotting. What factors would determine your choice of electrophoresis method ... 

Polyacrylamide gel (SDS-PAGE, IEF, native PAGE, or Tris-tricine SDS-PAGE) containing separated proteins (see Basic Protocols 1 and 2 see Alternate Protocols 1 to 4)... 

Native-PAGE (UNIT B3.1) can be used to assess the purity of the myoglobin extract, which should produce a single protein band with a molecular weight of 17.8 kDa. 

Another issue that should be considered carefully is the purity of nucleic acids used at each step. Since it is known that RNA can self-cleave and ligate, DNA produced from PCR that is entering the next cycle of selection should be size-purified by native PAGE (Section 8.3.1.5). Finally, all solutions should be prepared from DNase/RNase-free reagents in DEPC H20, 0.2 pm filtered, and stored at 4 °C (buffers) or —20 °C (especially nucleotide solutions). 

Probing the Function of Conformers Resolved by Native PAGE 201... 

This chapter will focus on the use of native PAGE to investigate folding of the Tetrahymena group I ribozyme. However, these protocols are easily adapted to other ribozymes and structured RNAs (e.g., Adilakshmi et ah, 2005 Lafontaine et ah, 2002 Pinard et ah, 2001 Severcan et ah, 2009). Detailed discussions of gel mobility shift methods for measuring protein—... 

A variety of commercial apparatuses or gel boxes for vertical PAGE are suitable for native PAGE experiments. Temperature control is critical to the separation of RNA conformers (slow exchange), and thus the box must be designed for use with a circulating water bath. We have obtained good... 

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