Gel analysis

Fig. 1. SDS gel analysis of proteins synthesised by excised maize roots incubated at continuous 40 °C. Roots of 3-day-old maize seedlings were excised and incubated at 40 °C for increasing times as indicated. Labelling with [ Sjmethionine was carried out in the final 20 min of the incubation. Proteins were visualised by fluorography. Mol wt distribution in kDa indicated at left. From Cooper Ho (1983).

Mailer, J. L., and Smith, D. S. (1985). Two-dimensional polyacrylamide gel analysis of changes in protein phosphorylation during maturation of Xenopus oocytes. Dev. Biol. 109 150-156. 

Joubert R et al. Two-dimensional gel analysis of the proteome of lager brewing yeasts. Yeast 2000 16 511-522. 

SDS polyacrylamide gel electrophoresis (SDS-PAGE) represents the most commonly used analytical technique in the assessment of final product purity (Figure 7.1). This technique is well established and easy to perform. It provides high-resolution separation of polypeptides on the basis of their molecular mass. Bands containing as little as 100 ng of protein can be visualized by staining the gel with dyes such as Coomassie blue. Subsequent gel analysis by scanning laser densitometry allows quantitative determination of the protein content of each band (thus allowing quantification of protein impurities in the product). 

A further improvement of the more traditional slab gel analysis is the use of high-performance capillary electrophoresis (HPCE), which combines the separation power of high-performance liquid chromatography (HPLC) with the selectivity and speed of conventional gel electrophoresis. However, as HPCE separations are often performed using fused silica capillaries the positively charged histone molecules... 

Mouse liver has been widely used for proteome analysis by the 2DE method. (7, 8) In this study, the extracted proteins from the mouse liver tissue are used as an example. Protein spots are evaluated using the gel analysis software ProFINDER 2D (Perk-inElmer Life And Analytical Sciences, Inc., Wellesley, MA) and PDQuest (Bio-Rad Laboratories, Hercules, CA)). 

In principle, detailed information about the radiation chemistry of a polymer may be obtained using sol-gel analysis as a function of dose to provide estimates of both crosslinks (x) and fractures (F) (5). Such estimates have been made in only one case for PET (18) but are supplemented by estimates made from other data obtained at high dose rates in Table II. The data used are scanty, generally only three points, and more detailed work is desirable. In addition, further work is required to... 

In 2001, both Lawrie et al. and Moskaluk coupled LCM (laser capture microdissection) with 2D gel proteomics to recover proteins from laser captured micro-dissected tissue in a form that can be used in 2D gel analysis and mass spectrometry. This may provide valuable information for protein profiling and databasing of human tissues in healthy and disease states. 

Mijnlibff, P. F., and W. J. M. Jaspers Thermodynamics of swelling of polymer-network gels. Analysis of excluded volume effects in polymer solutions and polymer networks. J. Polymer Sci. A-2 (in press). 

The solubility of proteins from parasitic organisms can often be enhanced by the use of hot sodium dodecyl sulphate (SDS) before solubilization in SD buffers. The ability of SDS to denature proteins also helps in allowing access to hydrophobic proteins, not normally seen after standard preparation procedures. SDS treatment in whole F. hepatica preparations has been shown to yield more protein spots visualized on gel analysis than other methods (Jefferies et a/., 2000). 

Bottaro, A., de Marchi, M., Migone, N., Carbonara, A.O. (1989). Pulsed-field gel analysis of human heavy-chain constant region deletions reveals the extent of unmapped regions within the locus. Genomics 4,505-508. 

The 1.2% agarose gel is prepared using agarose (usb) and 10 x TAE (Tris-Acetate-EDTA) pH 8.3 buffer (Invitrogen). Ethidium Bromide (Sigma) as marker and Low DNA Mass Ladder (Invitrogen) are used in agarose gel analysis. 

The second version of the post-gel analysis is equivalent to that of Stockmay-er [19]. He assumed that the distribution of sizes within sol past the gel point remains unchanged and the ratios of concentrations can still be deduced from those given in Table 5. Ziff and Stell [28] concluded that this assumption is equivalent to a disregard of reactions between gel and sol. Consequently, the concentration of each k-mer as well as that of the sol fraction decays according to the equation... 

Ziff and Stell [28] themselves proposed a third version of the post gel analysis which differed from Flory s one by disregarding the reactions taking place within gel molecule (no intramolecular reactions both in sol and gel). 

Fig. 1. IC-RT-PCR of potato latent virus. Agarose gel analysis of IC-RT-PCR assays from infected potato plants, immunocaptured using a PVS° polyclonal antibody, which also had affinity to potato latent virus (10). Lane M 100-bp molecular weight standard (SuperLadder-Low ABgene) lane 1, negative control (water) lane 2, positive control (potato virus S) and lane 3, potato latent virus.

Figure 4.3 Incorporation of PyC to position 75 in a tRNA transcript by CCA enzyme. (A) Chemical structure of PyCTP, where inward and outward arrows denote hydrogen bond acceptors and donors, respectively. (B) A scheme showing the cloverleaf of a tRNA-C74 transcript as the substrate for CCA addition, using PyCTP and ATP as the nucleotide donors. (C) Denaturing gel analysis (12% PAGE/7 M urea) of extension of the tRNA-C74 transcript by CCA enzyme, showing the tRNA transcript in lane 1, lack of extension of the transcript without CTP or PyCTP in lane 2, extension with polyC in lane 3, extension with C75—A76 in lane 4, extension with polyPyC in lane 5, and extension with PyC75-A76 in lane 6 (Zhang et al., 2008a).

We focus on position 75 to limit PyC incorporation to one well-defined location. In certain cases, it may be beneficial to incorporate PyC to both positions C74 and C75 in order to enhance the fluorescence emission signal of the probe. This is achieved by using a tRNA primer terminated at position 73 and by extending the primer with PyCTP and ATP as the nucleotide substrates. In the absence of normal CTP, complete extension of the primer from position 74 to 76, as visualized by denaturing gel analysis (e.g., Fig. 4.3C), is an indication that PyC has been incorporated at both positions. However, due to the rapid reaction of the CCA enzyme to synthesize consecutive C74 and C75 (Dupasquier et al., 2008), it would be difficult to incorporate PyC to just position 74. 

J Liao, N-O Ku, MB Omary. Two-dimensional gel analysis of glandular keratin intermediate filament phosphorylation. Electrophoresis 17 1671-1676, 1996. 

Steinberg RA, Coffino P. Two-dimensional gel analysis of cyclic AMP effects in cultured S49 mouse lymphoma cells protein modifications, inductions and repressions. Cell 1979 18 719-733. 

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