Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa

Surface expression of metabotropic glutamate receptor variants mGluR1a and mGluR1b in transfected HEK293 cells

“…After washing, cells were harvested and sonicated at 4°C in sample buffer (0.1 M Trizma-Base, 0.1 M NaCl, pH 7.4), and heated at 80°C for 15 min in treatment buffer (62,5 mM Trizma-Base, 2% Sodium-dodecyl sulfate (SDS), 10% glycerol, 190 mM Dithiothreitol (DTT), 0,01% bromophenol blue). Western blot analyses were carried out using 10% gels of total acrylamide in Tris/Tricine/SDS-PAGE system or in Tris/Glycine/SDS-PAGE (Laemmli, 1970;Schagger and von Jagow, 1987) µl SDS-PAGE sample buffer 80°C for 10min and 10µl were loaded on gels for immunoblot analysis.…”

Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants

“…Protein pellets were neutralized and dissolved in 15 ml 1 M Tris base plus 35 ml 2r sample buffer (100 mM Tris-HCl, pH 6.8, 4 mM EDTA, 4% SDS, 20% glycerol, 2% 2-mercaptoethanol, 0.02% bromophenol blue) and heated for 4 min at 95uC. Precipitated proteins (equivalent to 0.3 A 600nm lane) were separated by SDS-PAGE in 10% tricine gels (Schä gger and von Jagow, 1987 …”

A stable hairpin preceded by a short open reading frame promotes nonlinear ribosome migration on a synthetic mRNA leader

“…In vitro translation products were analyzed in an electrophoretic system (Schägger & von Jagow, 1987) that allows separation of the 4+6-and 7+9-kDa peptides derived from uORF F and the truncated CAT reporter gene, respectively (Fig+ 4A)+ An additional weak band migrating at the position of the expected uORF F product was indeed detected in the case of mutants with decreased stability within stem section I (pMH161, (Fig+ 2A) to replace the natural elongated hairpin structure of the wild-type leader+ All secondary structures used were based on published data demonstrating that an artificial stable hairpin can efficiently block 40S subunit scanning, even if not in direct proximity to the 59-terminal cap structure in the leader (Kozak, 1989a)+ Thus, hp7-type structures flanked by the CaMV 35S RNA leader terminal sequences were tested in translation in vivo and in vitro (Fig+ 3B)+ In both systems, replacement of the CaMV elongated hairpin structure by a short, low-energy stem, in either orientation (pMH188: Ϫ45 kcal/mol; pMH189: Ϫ46 kcal/mol), supports efficient translation, showing that the stem per se, and not its sequence, is recognized by the translation machinery+ Stability of the hair-FIGURE 3. Expression of the CAT-reporter gene under control of mutations in the leader+ A: Mutations in the 59 end (pMH176, pMH174, pMH119), 39 end (pMH150, pMH155, pMH164, pMH165, pMH167 pMH169, pMH177, pMH187)+ B,C: Structural mutations in stem section I (pMH175, pMH160, pMH158, pMH72, pMH163) and an artificial hairpin replacement in the CaMV 35S RNA leader (pMH188, pMH189, pMH191, pMH212, pMH200, pMH190)+ Mutations as depicted in Figures 2 and 5; expression levels in vivo and in vitro are represented by grey and black bars, respectively; resolution of in vitro translated products in 12% SDS-PAGE is shown in the lower panels; c in C indicates control with no external RNA included+ pin in pMH188 seems to be sufficient to support ribosome shunting, as extending the structure does not enhance translation further (Fig+ 3B; pMH190; Ϫ50 kcal/ mol)+ To analyze the mechanism of translation initiation on pMH188, we then introduced additional mutations that were previously found to affect translation under the control of the CaMV 35S RNA leader+ Deletion of uORF A and deletion of the complete 59-proximal unstructured region (Table 1; pMH191 and pMH212, respectively) seriously diminished translation when tested in the context of the pMH188 construct; deletion of most of the 39-proximal unstructured region (pMH200) had only a marginal effect on CAT expression (equivalent to constructs pMH174, pMH119, and pMH187 in the CaMV 35S RNA leader context, respectively; see Figs+ 2 and 5 for sequence of mutants)+ In vitro, as with constructs expressed under the control of the CaMV 35S RNA leader (Hemmings-Mieszczak et al+, 1998), translation on the pMH188 transcript is optimal at 30 8C and a potassium acetate concentration of 100 mM (Fig+ 6A)+ Altogether, the way the artificial low-energy stem supports translation in pMH188 appears similar to the translation mechanism occurring on the CaMV 35S RNA leader+ Generally, expression levels in vivo on the CaMV 35S RNA leader constructs parallel translation data in vitro indicating that our results reflect differences in translation efficiencies (Fig+ 3A)+ Constructs including artificial hairpins were expressed at a similar level in vivo as those under the control of the CaMV 35S RNA leader (Fig+ 3B); in in vitro experiments, however, their expression was slightly higher+ Differences in ionicstrength conditions between the two translation systems might be responsible for this effect+ In any case, when compared between themselves, all constructs with synthetic stems in the leader are expr...…”

“…CAT reporter gene expression under the influence of CaMV (strain S; Franck et al+, 1980) 35S RNA leader (all numberings are according to Hemmings-Mieszczak et al+, 1998) or artificial leader sequences was analyzed in vitro using pGEM-1-based constructs (Promega) or in transiently transfected Orychophragmus violaceus protoplasts using pDH51-based constructs (Pietrzak et al+, 1986)+ Preparation of expression constructs and introduction of additional restriction sites (Fig+ 2A; HindIII, XbaI, BamHI, EcoRI, Sal I, SpeI, and NcoI) were described previously (Hemmings-Mieszczak et al+, 1998)+ Mutations in the leader were introduced by pairs of annealed oligos subcloned between two relevant restriction sites+ All constructs were sequenced; sequences of oligonucleotides and constructs are available on request+ SP6-directed transcripts were synthesized on templates digested with Pst I or EcoRI (constructs used in Figs+ 4 and 6B did not contain EcoRI site in the leader) and used for in vitro translation assays giving the full-length or truncated CAT products, respectively+ In the former case, the in vitro translated products were separated in the 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); in the latter case the separation was performed in the 16+5% SDS-PAGE using the Tricine discontinuous buffer system (Schägger & von Jagow, 1987)+…”