Laser densitometry

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). 

Quantitate the radioactivity, as a measure of RT activity in 1 pL of the culture supernatant, either by liquid scintillation counting (cut out the spots and place them directly into scintillation fluid), by phosphorimage analysis and quantitation of relative pixel units, or by autoradiography and laser densitometry. 

Asmis, R., Buehler, E., Jelk, J., and Gey, K. F. (1997). Concurrent quantification of cellular cholesterol, cholesteryl esters and triglycerides in small biological samples. Reevaluation of thin layer chromatography using laser densitometry. J. Chromatogr., B Biomed. Sci. Appl. 691 59-66. 

Care to measure analytes at margins Is carried out by laser densitometry and allows visual dissection of zones. 

Estimation of spots Computerized laser densitometry and direct scintillation counting from plates defines geometry, exactly calculates ) /(peak), and quantifies zone. 

Bean, R and J.B. Peter (1994) Allelic D variants of transferrin in evaluation of alcohol abuse differential diagnosis by isoelectric focusing-immunoblotting-laser densitometry. Clin. Chem. 40 2078-2083. 

Laser densitometry, 940,941 Laser-pyrolysis scanning (LPS) for determination of pesticides, 811 Layer pre washing, 145 Layers. See Sorbents and layers Leather dyes. 1004,1017,1019-1020 Ligand exchange, TLC enantiomeric resolution via, 651-674 applications, 653-665... 

Spinach chloroplast envelope membranes were prepared according to [5]. Envelope protein samples (30 pg) were phosphorylated in the presence of 33 nM [y PjATP as described in [2]. The envelope protein kinase activities were stopped after incubation at 23°C for 1.5 min by adding one volume of SDS-PAGE sample buffer. The incorporated [ Pjphosphate into proteins was quantified by the Cerenkov procedure. Lipid extraction and thin layer chromatography (TLC) were carried out as described in [2] and [6]. Immediately after charring, the separated lipids on TLC plates were quantified by laser densitometry (Bioimage Millipore). 

Figure 4. Kinetics of the staurosporine-induced increase in tau protein levels.One day before the experiment, PC 12 cells were plated on 20-cm dishes coated with collagen/polylysine and treated with staurosporine (50 nM) or NGF (50 ng/ml) as indicated. After treatment, cells were harvested, lysed and subjected to electrophoresis on 10% SDS-PAGE, electrotransferred to nitrocellulose filters, and immunostained with anti-tau antibody and ECL detection system. (A) A representative Western blot autoradiograph of PC 12 cell cultures treated with staurosporine or NGF. Numbers on top of the autoradiograph indicate hours of treatment. Arrows indicate mobility of rainbow-colored molecular weight markers. (B) Quantitation of tau protein levels by laser densitometry in four different kinetic experiments. Levels of 55- and 110-kD tau proteins were analyzed separately. Values (mean standard error) represent tau proteins level in the treated cells relative to their level in unstimulated control cells.

Figure 5. Kinetics and dose-response of PMA induced increase in APP levels in PC 12 cells. PC 12 confluent cultures grown in 10 cm petri dishes coated with poly L-lysine and collagen have been exposed for 18 hours at 37°C to the indicated concentrations of PMA, lysed, submitted for SDS-PAGE and western blotting using a monoclonal antibody directed to the amino acids sequence 511-608 of APP 95. The level of APP protein in each sample was estimated by laser densitometry scanning and is represented as percentages of untreated cultures. Left insert represents a kinetic experiment performed under similar conditions using 200 nM PMA. Right insert represents a western experiment photograph of a PMA dose-response experiment. Arrows -position of the -100 kD APP protein, a -1 nM b - 20 nM c - 50 nM d - 100 nM e - 200 nM.

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