Non-denaturing PAGE

Fig. 11.1. Somatic and secreted AChEs in N. brasiliensis. Extracts of parasites collected 4 days (track 1) and 8 days (track 3) post-infection of rats were resolved by non-denaturing PAGE alongside secreted products from parasites also collected 4 days (track 2) and 8 days (track 4) postinfection, and AChE activity visualized by cytochemical staining. Secretory enzymes designated as forms A, B and C are indicated, and the non-secreted isoform is arrowed. Reproduced from Hussein etal. (1999b), with permission.

An antennal-specific aldehyde oxidase (AOX) of M. sexta (MsexAOX) was the next identified pheromone-degrading enzyme (Rybczynski el al., 1989). The activity of MsexAOX was visualized on non-denaturing PAGE, and was shown to be antennal specific but present in sensilla of both male and female antennae. MsexAOX was observed as a dimer with a combined estimated molecular mass of 295 kDa. M. sexta uses a multicomponent pheromone consisting exclusively of aldehydes including bombykal (Starratt el al., 1979 Tumlinson el al., 1989, 1994) MsexAOX was shown to degrade bombykal to its carboxylic acid. Both TLC and spectrophotometric assays were established and a variety of substrates and inhibitors were characterized. Making adjustments for the concentrations and volumes within a sensillum lumen, the in vivo half-life of pheromone was estimated at 0.6 msec in the presence of this enzyme (Rybczynski el al., 1989). 

Figure 2 Chaperonin- and Mg-ATP-dependent in vitro reconstitution of active dimeric Rubisco from denatured Rubisco. a) Rubisco activity, expressed as a percentage of activity observed with an equal quantity of native Rubisco. b) Western blot after non-denaturing PAGE of the reaction mixtures used in a and probed with antibody raised against Rubisco. c) as in b, but probed with antibody raised against cpn60. d) Summary of the reaction conditions. Reprinted by permission from NATURE vol. 342 pp. 884-889. Copyright (C) 1989 Macmillan Magazines Ltd.

Non-denaturing PAGE was carried out as described by Peter et al. (8). Pmified PS I isolated from sucrose adients was incubated at 37 C for 20 mins, and electrophoresed on a Deriphat non-denaturing gel. Denaturing SDS-PAGE was by a modification of the method of Laenu i (8), in ich 4M tirea was contained in the separating gel. 

Non-denaturing PAGE of the purified PS I complex yielded three pigment-protein bands (Fig.l). These pigment-protein complexes were subsequently identified as PS I, CCI and LHC I in order of decreasing size. CC I is blue-green, typical of other chi a and beta-carotene containing... 

Heme staining which identified both the intact complex in non-denaturing PAGE, or its dissociated subunits in SDS-PAGE Fig. 1. Fluorescence induction of y-1 and B6 in thylakoids from y-1 cells, did not react positively with... 

The properties of the chlorophyll-protein complexes containing non-phosphorylated polypeptides in mutant Uiylakoids could be different from those of y-1 cells. To test this possibility chlorophyll-protein complexes of in vitro phosphorylated membranes were resolved by non denaturing PAGE and their polypeptide composition and phosphorylation was detected by denaturing second dimension SDS-PAGE. 

Buffers containing SDS for cell lysis (Protocols lA and IB) are incompatible with some subsequent methods of analysis, for example non-denaturing PAGE (Protocol 12) or immunopredpitation under non-disruptive conditions (Protocol 14B). In order to study physiological interactions, it is necessary to perform cell lysis in buffers containing no detergents or mild non-ionic detergents, such as Triton X-100 or Nonidet P-40. 

Non-denaturing PAGE running buffen 25 mM Tris, 192 mM glycine pH 8.8... 

Assemble the glass plates and cast the non-denaturing PAGE gels using the mini gel system as described in Protocol 4. 

Add 1 jl1 of 10 X non-denaturing PAGE sample buffer to 10 pJ protein sample, mix well but do not heat. 

Clamp the gel to the mini gel apparatus and fill the upper and lower chambers with non-denaturing PAGE running buffer pre[Pg.289]

Connect the upper chamber to the cathode and the lower chamber to the anode and cany out non-denaturing PAGE at 4°C at constant voltage until the dye fi-ont reaches the bottom of the gel (typically 3 h at 90 V or overnight at 30 V). 

X non-denaturing PAGE sample buffer 125 iriM Tris-HCl pH 8.8, 84% (v ) glycerol, 0.1% (w/v) biomophenol blue... 

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