Gel blots

Fig. 2. Time course of accumulation of HSP mRNA. One jUg of poly(A) RNA isolated from soybean hypocotyls after different times of incubation at 42.5 °C (hs) or at additional times after transfer back to 28 °C after 4 h at the elevated temperature (recovery), were electrophoresed in formaldehyde agarose gels. Blots of these gels were hybridised with a mixture of four cDNAs encoding small soybean HSPs ranging from 15 to 23 kDa. From Schoffl Key (1982).

The amplification products were electrophoresed on agarose gels. Blots were performed according to protocols supplied with Hybond - N-nylon membrane (Amersham). 

B) RNA gel blot hybridization revealed massive enhancement of the PAP1 expression. Total RNA was isolated from 4-week-old papl-D and wildtype plants. 

RNA gel blot analysis of several members of the genus Papaver demonstrated that salutaridinol 7-O-acetyltransferase transcript accumulated in three-week-old seedlings of P. orientale and P. bracteatum, though not in P. atlanticum or P. nudicaule % P. orientale accumulates the alternate biosynthetic precursor oripavine, and P. bracteatum accumulates the morphine biosynthetic precursor... 

Close the clamp and place the cassette in gel blotting apparatus such that the membrane is close to the anode and the gel is close to the cathode. 

Fluorescence scanners resolve fluorescence as a function of spatial coordinates in two dimensions for macroscopic objects such as electrophoresis gels, blots and chromatograms... 

Autoradiography is the method of choice for detection and quantification of radioactive labeled samples in electrophoresis gels, blotting, or hybridization filters using X-ray films. The main drawback of X-ray films is a limited linear range of image density with respect to the amount of radioactivity. 

Two main types of analysis are required (a) qualitative determination of the presence of elements and (b) quantitative determination of the amount of elements or species of interest contained in pharmaceutical products. Most analyses for pharmaceutical applications involve separation steps combined with ICP-MS, such as HPLC-ICP-MS or gel electrophoresis and the analysis of gel blots by LA-ICP-MS. Phosphorylated proteins (e.g., (3-casein) have been measured by LA-ICP-MS with a detection limit of 16pmol. HPLC-ICP-MS has been employed for the identification and quantification of metabolites of bradykinin in human and rat plasma.1... 

Chemiluminescent probes enable highly sensitive quantitative analysis of proteins blotted from electrophoretic gels onto a supporting matrix. For a quantitative comparison, it is important to be able to correct for introduced variables such as antibody titre, temperature, substrate etc. Comparison of blots completed on different days requires a chemiluminescent standard. The situation is more complex with 2D gels, where only one sample per gel/blot is used. A method has been published for preparing a chemiluminescent standard for quantitative comparison of 2D Western blot (89). 

The same authors also examined the S-nitrosylation of proteins by nNOS, in neuronal tissue from mice (Figure 11.10). Conversion of nitrosylated to biotinylated cysteines was done as above. Biotinylation was, in this case, used to extract the (formerly) nitrosylated proteins from the total mixture of cellular proteins by selective binding to solid-phase attached streptavidin. After retrieving the bound material by reduction with excess free thiol, the samples were run on a gel, blotted, and individual proteins detected using an-... 

The bottom-up approach very much resembles classical protein identification strategies. The proteins in the proteome are first separated by 2D-GE (Ch. 17.3), or in some cases by SCX, size-exclusion (SEC), or affinity (AfC) chromatography. Specific proteins are excised from the gel, blotted, or electroeluted. The protein is digested, and the digest is analysed by LC-MS. The EC separation involves either RPLC with microcapillary or nano-LC columns (Ch. 17.5.2), or 2D-LC with typically SEC or SCX in the first dimension and RPLC in the second (Ch. 17.5.4). Alternatively, the sample may be introduced via either direct-infusion nano-ESl (Ch. 17.2), CE-MS (Ch. 17.5.6), or a microfluidic device coupled to MS (Ch. 17.5.5). 

RNA, if not too small, can also be detected by in-gel hybridization. Instead of denaturation/neutralization, the formaldehyde-containing gel is washed for 30 min in 0.1 M Tris-HCI (pH 7.5) (Wallace and Miyada, 1987). Ahmad et al. (1990) fractionated RNA in a formaldehyde-containing agarose gel in MOPS buffer (Section 9.1.3), washed the gel in DEPC-treated water (twice) and placed it between two sheets of gel blot paper (S S GB002) before drying in a gel dryer as described in the previous section. They hybridized with cDNA at 42°C in 50% formamide, 5 X Denhardt s solution and 5 X SSPE as described in Section 9.3.2. 

Both genes reach a peak in expression at around 13 DPF and then decline to very low levels by 25 DPF with HalO mRNA being somewhat more prevalent at 25 DPF than that of Ha5. Both are undetectable in dry seed by RNA gel blot analysis (data not shown). Since the gel blot method gave more accurate quantitative data we continued to use it for the analysis of the expression pattern of other genes of interest. 

Actin, Tubulin, and 3-Lipoxygenase have Similar Expression Patterns that are Different from those of SSP Genes. Table I summarizes the results of Northern gel blot analysis of polyA+RNA from embryos at 8, 13 and 25 DPF with respect to the expression of actin, tubulin, and B-LOX. The degree of expression of respective transcripts are reported as percent of maximum cpm of probe hybridizing to total RNA. Each of these genes shows the same general expression pattern although actin radioactivity declines more rapidly than that of tubulin, which in turn declines more rapidly than B-LOX. 

Figure 2. Northern gel blot of total RNA from staged embryos. Numbers indicate the relative cpm of probe bound (expressed as percent of maximum) at each stage of development for HaG5 and HaGlO.

No picture Not enough radioactivity Exposition time too brief Exposition temperature too high Bad contact between gel/blot/thin layer plate and film Gel is still moist Does the gel contain ENTRANCE and was the drying temperature above 70° C See Fig. 1.3. 

Moisture In gel/blot reduces the sensitivity. Diving temperature should lie between 65 and 70° C. 

Fig. 1. RNA gel blot of total RNA isolated from Brassica napus buds probed with the Arabidopsis A9 gene (4). The probe was labeled with digoxigenin by the polymerase chain reaction (Chapter 10). The RNA samples on the gel are bud size < I mm (lane 1), 1-2 mm (lane 2), 2-3 mm (lane 3), 3-4 mm (lane 4), 4-5 mm (lane 5), >5 mm (lane S). Each lane contains 10 Mg of total RNA. The exposure time was 30 min.

Fig. 5. Analysis of chioropiast DNA from streptomycin -spectinomycin resistant transformants for typical RFLP variation seen between recipient and donor sequences flanking the 16S rRNA gene. Total cell DNA was digested with Kpn I and Hind Ml (top) or Kpn I (bottom), the fragments separated on 1.0% agarose gels, blotted to nitrocellulose and probed with the labelled 1.4 kb Kpn-Hind fragment (top) located 3 to the 16S gene or the 2.0 kb Bam-Eco fragment (bottom) at the 5 end of the 16S gene (Fig. 4).

See also-. Capillary Electrophoresis Overview. Electrophoresis Principles Isotachophoresis Isoelectric Focusing Polyacrylamide Gels Two-Dimensional Gels Blotting Techniques. 

See alsa Electrophoresis Oven/iew Isoelectric Focusing Two-Dimensional Gels Blotting Techniques Proteins. Forensic Sciences DNA Profiling. Proteins Traditional Methods of Sequence Determination. Proteomics. 

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