Densitometric analysis

Colored substance zones were obtained which could be analyzed quantitatively. The (visual) detection limits were hypericin 1 ng, rutin and chlorogenic acid 5 ng, hyperoside — quercetin 10 ng per mm chromatogram zone. The detection limits for densitometric analysis are between 20 and 50% of those for visual detection. 

For any experiments involving the use of phosphospecific antisera, it is crucial to develop the blot (usually a separate one) with an antibody that detects the protein of interest irrespective of its state of phosphorylation. This loading control allows one to assess whether all lanes contain similar amounts of that protein. Ideally, the ratios of the signal strengths for the phospho and total antisera should be calculated from densitometric analysis of the blots. Care must be taken when doing this for proteins that resolve into multiple bands (dependent on their states of phosphorylation,... 

The extent of cell migration/invasion can be monitored by standard light microscopy and/or by densitometric analysis (e.g., using Image software). Normalize the extent of migration in each sample to the one of the corresponding internal control. 

One of the most widely used forms of gel electrophoresis is known as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyacrylamide gel has several advantages that account for its extensive use. It has minimal adsorptive properties and produces a negligible electroosmotic effect (Hjelmeland and Chrambach 1981). In identity tests, for the determination of molecular weight, SDS-PAGE has been shown to be an appropriate, fast, and easy method that is often used in quality control laboratories. The use of SDS-PAGE followed by a densitometric analysis, such as MS, is a helpful technique for the determination of peptide or... 

Evaluation of patient-derived data is performed by comparative densitometric analysis of ApoC-III IEF patterns from healthy controls (Fig. 4.5.10). 

Fig. 5.2.3 Agarose gel electrophoresis of a normal serum (top) and a serum from a type III dys-lipidemic patient (bottom), including the densitometric analysis of the two lanes (left and right, respectively). The normal serum sample shows an -fraction, which is clearly separated from the pre-/ - and the predominant -fraction. In the serum sample from a type III dyslipidemic patient, the pre-/ - and the -fraction are highly elevated and cannot be separated (therefore termed broad-j5-band), while the -fraction is reduced...

Fig. 3. (-)-Linalool does not affect the expression of p-Akt and Akt in the spinal cord of neuropathic mice. (A) Western blot analysis of lumbar spinal cord (L4—L5 segment) 7 days after SNL alone or in combination with linalool (100 mg/kg s.c.) or vehicle treatment Homogenates were from the ipsi(I)-and contra(C)-lateral side of the cord (pool of three animals for each experimental group). (B—D) Densitometric analysis of immunoblots probed with anti-Akt, anti-phospho-Akt (Ser473), and anti-GAPDH antibodies from three different experiments (mean SEM).

Fig. 3. Phosphorylation level of Akt and GSK-3/3 following focal cerebral ischemia. (A) Western blot analysis of phospho-Akt (Ser473) (p-Akt) and total Akt performed on brain cortical homogenates from rats sacrificed 24 h after MCAo shows a trend toward a decrease of p-Akt immunoreactivity in the ipsilateral (I), ischemic, cortex as compared to contralateral (C), nonischemic, side. This trend toward a reduction also occurred for total Akt expression, so that phosphorylation level (expressed as the ratio p-Akt/Akt) is not significantly affected by ischemia. The result is representative of three independent experiments. Histograms in (C) show the results of the densitometric analysis of the autoradioraphic bands corresponding to p-Akt, total Akt, and /3-actin. p-Akt and Akt levels were normalized to the values yielded by /3-actin and Akt phosphorylation was expressed by the ratio of p-Akt/total Akt data are reported as mean S.E.M. ( = 3 per group). The same samples were used for the subsequent western blot analysis of phospho-GSK-3/3 (Ser9) (p-GSK-3/3) and total GSK-3/3 and a representative result of three independent experiments is shown in (B). The results of the densitometric analysis of the bands corresponding to p-GSK-3/3, total GSK-3/3, and a-tubulin are reported in (C). p-GSK-3/3 and total GSK-3/3 were normalized to the values yielded by a-tubulin whereas GSK-3/3 phosphorylation was calculated from the ratio of p-GSK-3/3/total GSK-3/3 data are reported as mean S.E.M. (n = 3 per group).

Fig. 3. Changes in Akt and GSK-3/3 phosphorylation induced by transient retinal ischemia and effect ofintravitreal application of MK801 in rat. Animals were subjected to retinal ischemia for 50 min in the right eye (R) and reperfusion was allowed for 1, 6, or 24 h. The left eye (L) was used as control. (A) Phosphorylation of Akt on Ser473 is significantly diminished after retinal ischemia and is accompanied by a transient dephosphorylation (activation) of GSK-3/3. During the reperfusion phase, Akt activation is increased within 1 h whereas GSK-3(3 phosphorylation status returns to basal level. (B) Intravitreal treatment with MK801 enhances the phosphorylation of Akt reported after I h reperfusion. Histograms show the results of the densitometric analysis of immunoreactive bands. P < 0.05 versus vehicle.

The sample detection after electrophoresis can be quantified by a densitometric analysis of Coomassie brilliant blue stained spots. A silver stain is available to provide higher sensitivities when detection of samples in the nanogram range is required. 

Brozek, J., Grande, F., Anderson, J. T. Keys, A. (1963) Densitometric analysis of body composition revision of some quantitative assumptions. Ann. N.Y. Acad. Sci. 110, 113-140. 

Figure 4-22. Densitometric analysis of serum separation. When electrophoresis is complete, the cellulose acetate film is removed and stained. Excess dye, not bound to protein, is removed by destaining and the film is transferred to a bath in which the cellulose acetate becomes transparent. The film is placed in a scanner in which it is moved across the light path of a spectrophotometer recording the absorbance of the blue stained protein peaks. The result is a densitometric scan whose peaks can be integrated to quantitate the protein profile of the serum.

As measured by densitometric analysis after SDS-PAGE. h ercentage of total protein expressed under a particular growth condition. Total amount expressed relative to Trp. 

In some cases, such as that of gelatinases, the method not only reveals the activated form of the enzyme but also the zymogen, giving rise to a closely spaced doublet of digestion. This counterintuitive outcome results from the electrophoresis conditions, which may expose binding and active sites of the enzyme without loss of the propeptide. Densitometric analysis of each band of the doublet may then be used to measure the activation level of a protease. 

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