Dot-blot analysis

M.S. Ristaldi, M. Pirastu, C. Rosatelli, G. Monni, H. Erlich, R. Saiki, A. Cao, Prenatal Diagnosis of Beta-thalassaemia in Mediterranean Populations by Dot Blot Analysis with DNA Amplification and Allele Specific Oligonucleotide Probes , Prenat. Diagn, 9(9), 629-638 (1989). 

Optimal dilution of the primary and secondary antibody should be determined by immunoblotting of one-dimensional gels. Dot blot analysis can also be used. Working solutions of antibodies can be stored at -20°C and used several times (82). 

Fig. 2.4. Vector characterization. (A). SDS acrylamide gel electrophoresis of a rAAV-UF-11 vector. Approximately 2.5 x 1011 drp were loaded on a 10% SDS-polyacrylamide gel. The three capsid protein bands in the correct stoichiometry are visualized by silver staining. (B) Infectious center assay of a rAAV-CB-hAAT stock when probed for the transgene each spot represents a vector infected cell. (C) Dot blot analysis of the rAAV stock to determine the titer of DNase-resistant particles of rAAV. Amounts (ng) refer to a serial dilution of the packaged rAAV vector plasmid used to construct the standard curve. The stock has a calculated titer of 4.3 x 1013 drp/ml.

Fig. 3. Activity of HRP following LEF induced uptake into COS 5-7 and HaCaT cells. Cells (2 x 106/mL) were exposed to LEF (20 V/cm, 500 Hz, and pulse width 180 (is) for 1 min in the presence of 1 mg/mL HRP in DMEM-H medium. Three hours after exposure, the cells were washed three times in PBS and disrupted by five freeze-thaw cycles in 50 (jlL PBS containing anti-protease cocktail (1 100). Dot-blot analysis of HRP activity 1 - cells without HRP 2 - non-exposed cells with HRP 3 and 4 -duplicates of cells exposed to LEF in the presence of HRP.

Other studies have reported expression of CYPs in human stomach mucosa. The recently identified CYP2S1 mRNA was detected by dot blot analysis (24), and CYP2J2 was detected by immunoblot (99). 

If the plasmid has not been radiolabeled, the amount of DNA taken up following transfection can be evaluated using dot-blot analysis (see Note 23). 

Figure 1. Dot blot analysis of HaG5 and HaGlO expression. One pg of total RNA from embryos at the indicated day post-fertilization (DPF) was bound to each dot on nitrocellulose and the dot blots were then hybridized to the appropriate probe, washed and exposed to film overnight. "Dry" - mature dry seed, Germ. germinating dry seed.

Because dot blot analysis allows for more samples to be processed on a single membrane and because dot blots are more easily quantified this technique was also used to assess HCP clearance during DSP. An example is shown in Fig. 2. As can be seen from this figure dot blotting can be performed with reasonable reproducibility and sensitivity. The lowest concentration of the calibrator that can be quantified reliably is about 100 ng/mL. This allows for dilution of 2.1 (6,000-50,000) and 3.1 samples (200-2,000) while 5.1 samples Were run undiluted. To obtain reliable results samples were diluted using sample weights instead of sample volumes. Within day reproducibility was better than 10% while day to day reproducibility was better than 30%. 

Fig. 2. Dot blot analysis (top left) Dot blot stained with HCP polyclonal antibodies. The top two rows are calibration standards of HCP mix in duplicate (7 dilutions from 3.8-0.19 pg/mL plus blank, from left to right). The third and fourth rows are 8 dilutions in duplicate (48,000-6,000, left to right) of a 2.1 1PV production fraction sample while the fifth and sixth rows are 8 dilutions in duplicate (2,000-250, left to right) of a 3.1 1PV production fraction. Corresponding profile data of the dot blot are shown on the right with the calibration curve (bottom left)...

Figure 5-2 Expression of hemopexin mRNA levels in barrier tissues. The data show the results of a dot blot analysis of various amounts of mouse liver (0.2, 0.5,0.8, 1.0 and 2.0 pg), brain (10 and 20 pg), ovary (5 and lOpg), testis (10 and 20pg) RNA samples (Ambion, Texas, USA) as indicated and the levels of mRNA were determined by hybridization with the same hemopexin probe as described in the legend to Figure 5-1.

Figure 9. Cutinase mRNA content from spores of F. solani f. sp. pisi exposed to either cutin hydrolysate or purified dihydroxy-Cx acid for various periods of time. Cutinase [ P]cDNA was used as a probe in the dot blot analysis. (Reproduced with permission from Ref. 35. Copyright 1986 The National Academy of Sciences.)...

Perform immuno-dot blot analysis (described in Subheading 3.3.2.) of chromatography fractions to determine PIA-containing samples. Most PIA elutes shortly after the exclusion volume. 

Dot Blot Analysis to Estimate GAG Quantity/Concentration 2.3.1 Required Materials... 

It is not straightforward to measure concentrations and amounts of GAG preparations, which are not enzymatically treated and are yet unfractionated. One of the easiest and quickest methods is the so-called dot blot analysis. For this purpose, 2 pL of standard concentrations (heparan sulfate) ranging from 2.5 to 25 pg are spotted onto positively charged nylon transfer membrane. Further spot three times 2 pL and five times 2 pL of your samples onto the membrane so that you have a spot with a total of 6 pL and a spot with 10 pL. Let the spots dry in between the spottings and after the last spot for at least 10-15 min. Perform an azure A (0.05% azure A in 5% acetic acid) stain, destain with water, and perform densitometry analysis of your samples. Calculate the concentration by comparing to the standard curve obtained with the HS standard. 

A Tau cDNA probe, prepared from an inmature brain cDNA library, hybridizes with Tau iriRNAs of 6 kb encoding both for juvenile and mature Taus (29). This probe was therefore used (30) to quantitate both iinnature and adult Tau niRNAs during brain development. Northern and dot blot analysis showed that the abundance of Tau iriRNA doubles from a late fetal stage (-4 days) until birth, remains constant until day 6 postnatal and... 

Fig. 2. Genotyping mice by dot-blot analysis. Tail DNA from intecross litters carrying a single-copy gene trap insertion wwe hybridized to a lacZ probe. Dots showing weak signals represent heterozygous animals, whereas those showing signals of twice the intensity represent homozygotes.

Figure 4.7 Dot-blot analysis of the ability of the silica nanoparticles to bind proteins. Nanoparticles dotted on glass slides were...

Fig. 2 Increasing content recovery by coupling luciferase-based assays with high-throughput biochemical readouts. The same cell line subjected to the luciferase-based assay protocol (HCT116 cells) is evaluated here for its reliability In reporting a biochemical readout (p53 expression) by dot blot analysis. Cells transfected with indicated expression construct and pathway reporters in a 96-well culture plates were lysed 48 h posttransfection. Following luciferase activity measurements (data not shown), protein from spent lysates was immobilized on nitrocellulose using a liquid handler and filtration manifold, (a) p53 and p-actin protein levels detected using protein-specific antibodies, infrared fluorescent dye-coupled secondary antibodies (with emissions at 680 and 800 nM), and the Li-COR imaging system. Columns of lysate corresponding to cells transfected with p53 DNA are boxed, (b) Quantification of the p53 to p-actin protein ratio...

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