Native PAGE eliminates the problem of PEG-SDS interaction in SDS-PAGE and provides an alternative to HPLC in characterization of protein PEGylation

PEGylation of proteins has become an increasingly important technology in recent years. However, determination and characterization of the PEGylation products are problematic especially for the reaction mixture containing various modified proteins, unreacted PEG, and unmodified protein. A comparative study was carried out with two HPLC methods and two electrophoresis methods for characterization of the reaction mixture in PEGylation of HSA with PEG 5000, 10000, and 20000. RP-HPLC fails to give the correct information about the reaction of PEG 20000. Size-exclusion HPLC (SE-HPLC) produced very poor resolution on the PEG 5000 reaction. SDS-PAGE can run multiple samples of all PEGylation but the bands were smeared or broadened probably due to the interaction between PEG and SDS. On the other hand, native PAGE eliminates the problem of PEG-SDS interaction and provides better resolutions for all samples. Various PEGylated products and unmodified protein migrate differentially in native PAGE under nondenatured conditions. The results demonstrated that native PAGE could be a good alternative to HPLC and SDS-PAGE for the analysis of PEG-protein conjugates especially for characterization of the PEGylation mixture.     

Analysis and purification of peptide nucleic acids by denaturing PAGE

A flexible and convenient protocol for the analysis and purification of peptide nucleic acid (PNA) oligomers and PNA-peptide chimeras by denaturing PAGE is described. Vertical slab gel electrophoresis, 26% in polyacrylamide and 8 M urea at pH 3, was suitable for analysis of oligomers ranging in size from tetramers (4-mers) to tetradodecamers (24-mers). Single-base resolution of oligomers was achieved and separations are generally superior to those given by standard RP-HPLC techniques. The separation of a related series of PNA oligomers showed the distance migrated was linearly dependent on the logarithm of the molecular weight. The migration of oligomers through the gel is dependent on the number of basic functional groups present, such as amino groups, and the A and C content of the oligomer. PNAs are amenable to detection by UV-shadowing technique illuminated at 260 nm or Coomassie blue staining, both with similar, sub-microgram per band detection limits.     

Preparative alkaline urea gradient PAGE: application to purification of extraordinarily-stable disulfide-linked homodimer of human growth hormone

The 40-60 pituitary human growth hormone (hGH) isoforms are so similar in their physico-chemical properties (charge, size, hydrophobicity) that the limited resolutions of chromatographic separation methodologies have not permitted most of them to be isolated. However, application of high-resolution preparative alkaline urea gradient PAGE has facilitated isolation of a disulfide-linked mercaptoethanol-resistant (MER) 45 kDa hGH dimer. Human pituitary extracts were separated by Sephadex G-100 chromatography under alkaline conditions. Pooled fractions containing MER-45 kDa hGH, as determined by SDS-PAGE, were then separated by Sephadex G-100 chromatography under acidic conditions followed by diethylaminoethyl (DEAE) anion-exchange chromatography. Pooled DEAE fractions containing MER-45 kDa hGH and other hGH isoforms were then separated by preparative electrophoresis in an alkaline polyacrylamide gradient (5-20%) slab gel containing 8 M urea into five distinct protein zones. One electroeluted zone contained pure MER-45 kDa hGH. The dimeric hGH isoform was immunoreactive at low concentrations (effective dose to produce 50% response (ED(50)) +/- S.E. = 58 +/- 5.00 pM) in a hGH radioimmunoassay, similar to that of standard monomeric hGH (ED(50) +/- S.E. = 22.93 +/- 3.90 pM), indicating that is was conformationally intact. Alkaline urea gradient PAGE is a valuable tool for preparative separation of structurally similar proteins such as isoforms of the hGH family.     

Histochemical staining and quantification of dihydrolipoamide dehydrogenase diaphorase activity using blue native PAGE

Mammalian mitochondrial dihydrolipoamide dehydrogenase (DLDH, EC 1.8.1.4) catalyzes NAD(+)-dependent oxidation of dihydrolipoamide in vivo and can also act as a diaphorase catalyzing in vitro nicotinamide adenine dinucleotide (reduced form) (NADH)-dependent reduction of electron-accepting molecules such as ubiquinone and nitroblue tetrazolium (NBT). In this paper, we report a gel-based method for histochemical staining and quantification of DLDH diaphorase activity using blue native PAGE (BN-PAGE). Rat brain mitochondrial extracts, used as the source of DLDH, were resolved by nongradient BN-PAGE (9%), which was followed by diaphorase activity staining using NADH as the electron donor and NBT as the electron acceptor. It was shown that activity staining of DLDH diaphorase was both protein amount- and time-dependent. Moreover, this in-gel activity-staining method was demonstrated to be in good agreement with the conventional spectrophotometric method that measures DLDH dehydrogenase activity using dihydrolipoamide as the substrate. The method was applied to determine levels of DLDH diaphorase activity in several rat tissues other than the brain, and the results indicated a similar level of DLDH diaphorase activity for all the tissues examined. Finally, the effects of thiol-reactive reagents such as N-ethylmaleimide (NEM) and nitric oxide donors on DLDH diaphorase activity were evaluated, demonstrating that, with this method, DLDH diaphorase activity can be determined without having to remove these thiol-reactive reagents that may otherwise interfere with spectrophotometric measurement of DLDH dehydrogenase activity. The gel-based method can also be used as a means to isolate mitochondrial DLDH that is to be analyzed by mass spectral techniques in studying DLDH post-translational modifications.     

A comparison of Tris-glycine and Tris-tricine buffers for the electrophoretic separation of major serum proteins

This paper compares different buffer systems for the electrophoretic separation of the five most abundant serum proteins on native-PAGE gel and cellulose membranes. A modified Tris-tricine system was shown to be superior for the separation of these serum proteins in a 7% m/v native-PAGE gel as compared with the traditionally used Tris-glycine and Tris-tricine methods. This modified Tris-tricine buffer system was also employed for the separation of serum proteins using a cellulose acetate membrane and very effective separation was observed as compared with the traditionally used Tris-barbital and Tris-glycine buffer systems.     

Protocol for high-resolution electrophoresis separation of myosin heavy chain isoforms in bovine skeletal muscle

In this short communication we describe a specific protocol for SDS-PAGE separation of adult bovine myosin heavy-chain (MyHC) isoforms. The conditions defined in this protocol allow a good separation with a good reproducibility of the four MyHC isoforms (MyHC I, IIa, IIx, IIb) identified in adult skeletal muscle of this species. This procedure uses mini-gel electrophoresis system and does not involve preparation of gradient separating gels. In addition, this protocol can also be applied to the electrophoretic separation of ovine and camel MyHC isoforms.     

Blue native/SDS‐PAGE combined with iTRAQ analysis reveals advanced glycation end‐product‐induced changes of synaptosome proteins in C57 BL/6 mice

Evidence shows that administration of high‐level D ‐galactose induces the production of advanced glycation end‐products (AGEs) that have been implicated in the development of diabetic complications such as neuropathy. The deterioration of learning and memory during neuropathy might be associated with the altered expression of proteins in synapse. To evaluate AGE‐induced protein network alterations in synapse, blue native/SDS‐PAGE and iTRAQ proteomic methods were used to screen for differentially expressed synaptic proteins of cerebral cortex in D ‐galactose‐induced C57 BL/6 mice. In total, the expression level of 84 proteins is changed during AGE accumulation. The significantly differentially expressed proteins mainly participate in neurotransmission, energy metabolism and signal transduction pathway, suggesting that energy metabolism is damaged and neurotransmission is attenuated in synapse. The results of in vivo activities of malondialdehyde and superoxide dismutase suggested that AGE accumulation in the brain leads to the generation of reactive oxygen species. Therefore, elucidating the differentially expressed proteins underlying the AGE accumulation will open a new window to the mechanism of learning and memory impairments in neuropathy.     

Short-chain polyphosphates: Extraction effects on migration and size estimation of Saccharomyces cerevisiae extracts with polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis is commonly used to characterize the chain length of polyphosphates (polyP), more generally called condensed phosphates. After separation, nonradioactive, optical polyP staining is limited to chain lengths greater than 15 ${\mathrm{PO}}_3^{-}$monomers with toluidine blue or 4′,6-diamidino-2-phenylindole. PolyP chain lengths longer than 62${\mathrm{PO}}_3^{-}$monomers were correlated to the shortest DNA ladders. In this study, synthetic linear polyPs (Sigma-Aldrich “Type 45”, estimated mean length of 45 ${\mathrm{PO}}_3^{-}$ monomers), trimetaphosphate (trimetaP: 3 ${\mathrm{PO}}_3^{-}$ ring), tripolyphosphate (tripolyP), pyrophosphate (PP_i), and inorganic orthophosphate (o-P_i) were visualized after separation by an in situ hydrolytic degradation process to o-P_i that was subsequently stained with methyl green. Statistically insignificant migration reduction of synthetic short-chain polyP after perchloric acid or phenol–chloroform extraction was confirmed with the Friedman test. ³¹P diffusion–ordered NMR spectroscopy confirmed that extraction also reduced PP_i diffusivity by <10%. Linear regression between the R_f peak migration value and the logarithm of synthetic polyP molecular weights enabled estimation of extracted polyP chain lengths from 2 to 45 ${\mathrm{PO}}_3^{-}$monomers. Linear polyP extracts from Saccharomyces cerevisiae grown in aerobic conditions were generally shorter than extracts cultured in anaerobic conditions. Extractions from both aerobic and anaerobic S. cerevisiae included tripolyP and o-P_i, but no PP_i.