Quantifying Western blots: Pitfalls of densitometry

Although Western blots are frequently quantified, densitometry is not documented and appears to be based merely on traditions and guesswork. Confirming previous experience, none of 100 randomly selected and systematically scanned most recent papers provided sufficient information on how Western blot results were translated into statistical values. The importance of such information, however, becomes evident from our correlations of plasma erythropoietin values of various mammals determined using RIA and Western blot densitometry. Different common densitometry procedures applied to the identical Western blot revealed p‐values of these correlations ranging from 0.000013 to 0.76 reflecting the necessity of a scientifically sound basis for densitometry of Western blots. At present, the current lack of any definitions in densitometry opens the door to uncontrollable acquisition of any desired p‐value. Here we provide data that define what should be considered, what avoided and what documented when quantifying Western blots.     

Soft-laser scanning densitometry performed on a 35-mm slide illustrating SDS-PAGE separation of adenovirion polypeptides

Virion polypeptides (35 S), methionine-labeled and purified by CsCl gradient centrifugation, were separated by SDS-gel electrophoresis. Analysis of their band pattern was performed by scanning the images of the SDS-gels shown on a 35-mm slide. The densitometric tracings revealed the presence of 17 protein bands, although only 15 of them were visible to the naked eye. The high sensitivity and resolving capacities of the soft-laser scanning densitometer enabled us to detect trace amounts of protein bands separated in SDS-gels and to obtain a resolution compatible to that of electrophoresis. Fourfold electronic amplification of the densitometric tracings, produced by a computer, generated new information regarding the pattern of the electrophoregram. The facility to amplify peaks of importance is particularly advantageous when faint or overlapping protein bands revealed on a gel are assessed.     

Use of reflectance in soft laser densitometry in scanning opaque electrophoregrams

The present report describes a new approach in soft laser scanning densitometry performed on opaque electrophoregrams. The method employs a light reflectance system instead of absorption, which is commonly used. The data of this preliminary study were obtained by directly scanning a photograph of an electrophoregram exhibiting viral proteins separated by SDS-PAGE. Comparison of the two methods clearly demonstrate that laser reflectance offers finer resolution than laser absorbance when opaque materials are scanned.     

Dilatometric and calorimetric studies of the effect of staphylococcus aureus delta-lysin on the phospholipid phase transition

The interaction of the protein -lysin from Staphylococcus aureus with model membrane of dipalmitol-sn-glycero-3-phosphocholine has been studied by the method of temperature scanning densitometry and high precision differential scanning calorimetry. The studies were performed in the range of low relative protein concentrations (between 10^–4 and 3×10^–2 moles -lysin per mole phospholipid) in order to observe those effects which may be relevant to the high efficiency of membranolysis. -lysin was found to have strong effects on the main transition as well as on the pretransition, i.e., significant reduction of the transition volume and transition enthalpy and complete abolition of the pretransition. The temperature of the main transition was nearly unaffected in this concentration range. These effects can be interpreted as long-range structural changes in the lipid bilayers caused by the protein and are discussed in terms of a cooperative cluster model. The results are in many respects qualitatively similar to those observed earlier with the bee venom protein melittin.Presented at the sixth Italian meeting on Calorimetry and Thermal Analysis (AICAT) held in Naples, December 4–7, 1984     

Fluorescence detection by intensity changes for high-performance thin-layer chromatography separation of lipids using automated multiple development

Changes in emission of berberine cation, induced by non-covalent interactions with lipids on silica gel plates, can be used for detecting and quantifying lipids using fluorescence scanning densitometry in HPTLC analysis. This procedure, referred to as fluorescence detection by intensity changes (FDIC) has been used here in combination with automated multiple development (HPTLC/AMD), a gradient-based separation HPTLC technique, for separating, detecting and quantifying lipids from different families. Three different HPTLC/AMD gradient schemes have been developed for separating: neutral lipid families and steryl glycosides; different sphingolipids; and sphingosine-sphinganine mixtures. Fluorescent molar responses of studied lipids, and differences in response among different lipid families have been rationalized in the light of a previously proposed model of FDIC response, which is based on ion-induced dipole interactions between the fluorophore and the analyte. Likewise, computational calculations using molecular mechanics have also been a complementary useful tool to explain high FDIC responses of cholesteryl and steryl-derivatives, and moderate responses of sphingolipids. An explanation for the high FDIC response of cholesterol, whose limit of detection (LOD) is 5 ng, has been proposed. Advantages and limitations of FDIC application have also been discussed.     

Different stability‐indicating chromatographic methods for specific determination of paracetamol,dantrolene sodium,their toxic impurities and degradation products

A well‐known analgesic (paracetamol, PAR) and skeletal muscle relaxant [dantrolene sodium (DNS)] have been analyzed without interference from their toxic impurities and degradation products. The studied PAR impurities are the genotoxic and nephrotoxic p‐amino phenol (PAP) and the hepatotoxic and nephrotoxic chloroacetanilide, while 5‐(4‐nitrophenyl)‐2‐furaldehyde is reported to be a mutagenic and carcinogenic degradation product of DNS. The five studied components were determined and quantified by TLC–densitometric and RP‐HPLC methods. TLC–densitometry (method 1) used TLC silica gel and chloroform–ethyl acetate–acetic acid–triethylamine (7:3:0.5:0.05, by volume) as the mobile phase with UV scanning at 230 nm, while RP‐HPLC (method 2) was based on separation on a C₁₈ column using methanol–water (55:45, v/v pH 3 with aqueous formic acid) as mobile phase at 1 mL/min and detection at 230 nm. The developed methods were used for determination and quantification of the five studied components in different laboratory‐prepared mixtures. The were also applied for analysis of Dantrelax^® compound capsules where no interference among the studied components with each other or from excipients was observed. The methods were validated as per International Conference on Harmonization guidelines, and they compared favorably with the reported ones.     

Improved separation and quantitative determination of hydrocarbon types in gas oil by normal phase high‐performance TLC with UV and fluorescence scanning densitometry

Improved methods for separation and quantitative determination of hydrocarbon types from gas oil have been developed, which were based on high‐performance thin‐layer chromatography with ultraviolet and fluorescence scanning densitometry using horizontal elution. One of the methods allows the separation, detection, and determination of alkanes and naphthenes to be carried out, using berberine‐impregnated silica gel HPTLC plates, elution with n‐hexane, and berberine‐induced fluorescence detection at 365 nm. Another developed method allows total aromatics to be determined using silica gel HPTLC plates by elution with n‐hexane and acetone, and UV detection. In turn, PACs over three aromatic rings can be determined on either silica gel or caffeine‐impregnated silica gel HPTLC plates, elution with n‐hexane, and selective detection using native fluorescence at 365 nm. Concentrations lower than 5 wt% can be determined using this technique. In addition, a technique for an efficient, baseline‐resolved separation of a gas oil according to the number of aromatics rings (mono + di‐, tri‐, and polyaromatic compounds with more than three rings) is presented here. This technique involves a multistep elution on a mixed (silica gel and caffeine‐impregnated silica gel HPTLC plate) using a counter‐elution device, and UV detection.     

Temperature‐dependent polymorphic crystalline structure and melting behavior of poly(butylene adipate) investigated by time‐resolved FTIR spectroscopy

The polymorphic crystalline structure and melting behavior of biodegradable poly(butylene adipate) (PBA) samples melt‐crystallized at different crystallization temperatures were studied by differential scanning calorimetry (DSC) and fourier transform infrared (FTIR) spectroscopy. The crystalline structure and melting behavior of PBA were found to be greatly dependent on the crystallization temperature. By comparison of the FTIR spectra and the corresponding second derivatives between the α‐ and β‐crystal of PBA, the spectral differences were identified for the IR bands appeared at 1485, 1271, 1183, and 930 cm^?1 and the possible reasons were presented. Especially, the 930 cm^?1 band was found to be a characteristic band for the β‐crystal. Combining the DSC data with the analysis of normalized intensity changes of several main IR bands during the melting process, the melting behaviors of the α‐ and β‐crystal were clarified in detail. It is demonstrated by the in situ IR measurement that the β‐crystalline phase would transform into the α‐crystalline phase during the melting process, and the solid–solid phase transition from the β‐ to α‐crystal was well elucidated by comparing the intensity changes of the 1170 and 930 cm^?1 bands. The dependence of the β‐ to α‐crystal phase transition on the heating rate was revealed by monitoring the intensity ratio of the 909 and 930 cm^?1 band. It was suggested that at the heating rate of 0.5 or 1 °C/min, the percent amount of the transformed α‐crystal from the β‐crystal was much higher than that at the higher heating rate. The β‐crystal transforms into the α‐crystal incompletely at the higher heating rate because of the less time available for the phase transition. In addition, the β‐ to α‐crystal phase transition was further confirmed by the IR band shifts during the melting process. © 2009 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 47: 1997–2007, 2009     

Filling‐level effect on the physical properties of MgBr2‐ and MgCl2‐filled poly(vinyl acetate) films

The structural properties of poly(vinyl acetate) (PVAc) films filled with different levels of MgBr₂ or MgCl₂ were investigated. Differential scanning calorimetry revealed that, at certain filling levels of MgBr₂ or MgCl₂, two new transitions appeared. The first one was due to the α relaxation (T_α) associated with a crystalline region, and the second was due to the melting temperature (T_m). This implies that a crystalline phase was formed in the polymeric matrix. Changes occurring in T_α, T_m, and the melting peak area as a function of the filling level were examined. The X‐ray diffraction (XRD) pattern revealed that the pure PVAc film was amorphous. However, the addition of MgBr₂ or MgCl₂ led to the formation of a crystalline phase in the polymeric matrix that depended on the filling level. XRD also demonstrated that the structural changes depended not only on the metal ions but also on the halide ions. Scanning electron microscopy images for the studied samples were examined. © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 112–119, 2003     

Quantitative determination of phospholipids in a pharmaceutical drug by scanning and video densitometry

This paper describes a convenient and practice method for quantitation of surfactant phospholipids (1,2-dipalmitoyl-3-sn-phosphatidyl choline [DPPC] and 1-palmitayl-2-oleyl-3-sn-phosphatidyl glycerol [POPG]) in a recombinant surfactant lyophile (Venticute) by high-performance, thin-layer chromatography (HPTLC) with video densitometry. DPPC and POPG were extracted from Venticute-lyophile using methanol. Separation from the other active ingredients and excipients was accomplished by HPTLC on silica gel F254 plates with a mixture of chloroform, methanol, glacial acetic acid, and water as development solvent. Postchromatographic derivatization by dipping in copper sulphate/phosphoric acid reagent and subsequent heating shows grey-brown bands on a light blue background. These were detected with the video densitometer in the VIS range, and with scanning densitometry at 365 nm. Linear calibration in a working range of 0.7-1.3 microg DPPC and 0.35-0.65 microg POPG was demonstrated by integrating the area under the peaks. Good results were obtained with recovery experiments. When compared to classical slit scanning densitometry, video densitometry represents a fast alternative to quantitate thin-layer chromatograms in surfactant phospholipid analysis.     

Correlation of TLC/densitometry-data with capillary GC-data of some main volatile components of essential oils

The results of the densitometric and the capillary gas chromatographic investigations of numerous lavender oils of different origin and of essential oils of the woody group were correlated. Especially the main components, the monoterpenes linalool (¯1) and linalyl acetate (¯2), were quantified by densitometry with an average standard deviation of 6–7% in the examined oils. The use of TLC in combination with densitometry in the quantification of the main components to essential oils is thus a valuable addition to capillary gas chromatographic investigation. In addition to this, a useful and efficient alternative is given to groups specialized in TLC/densitometry to obtain data on such volatile compounds of essential oils during quantification. The use of TLC/densitometry thus combines the advantage of LC-with detection of also less volatile compounds—and the possibility of the parallel investigation of more samples in one step in a very efficient way.     

Determination of platinum, palladium and rhodium in Pt-catalysts by high performance thin layer chromatography using densitometry

The determination of palladium and platinum in alumina-supported bimetallic Pt-Re, Pd and Pt catalysts at the 0.03 and 0.3% (m/m) levels, respectively, and of rhodium and platinum in platinum sieve catalysts at the 0.04% (m/m) level and in the 4.75–19.75% (m/m) range, respectively, is described. The platinum group metals in fresh or spent catalysts are determined chromatographically using densitometry by forming theirN,N-diethyl-N-benzoylthiourea complexes. The precision of the method, %RSD, is 0.4–3.5, 2.2–6.7, and 4.2–6.0 for Pt, Pd and Rh, respectively. The accuracy was tested using alumina-based and active carbon platinum and palladium standards. The results were also compared with those obtained by FAAS. There was at most 8% difference between the results obtained with these methods, except for one active carbon based Pd standard.     

Densitometry and indirect normal‐phase HPTLC–ESI–MS for separation and quantitation of drugs and their glucuronide metabolites from plasma

The present work describes novel methods using densitometry and indirect or off‐line high performance thin‐layer chromatography–mass spectrometry (HPTLC–MS) for the simultaneous detection and quantification of asenapine, propranolol and telmisartan and their phase II glucuronide metabolites. After chromatographic separation of the drugs and their metabolites the analytes were scraped, extracted in methanol and concentrated prior to mass spectrometric analysis. Different combinations of toluene and methanol–ethanol–n‐butanol–iso‐propanol were tested for analyte separation and the best results were obtained using toluene–methanol–ammonia (6.9:3.0:0.1, v/v/v) as the elution solvent. All of the drug–metabolite pairs were separated with a homologous retardation factor difference of ≥22. The conventional densitometric approach was also studied and the method performances were compared. Both of the approaches were validated following the International Conference on Harmonization guidelines, and applied to spiked human plasma samples. The major advantage of the TLC–MS approach is that it can provide much lower limits of detection (1.98–5.83 pg/band) and limit of quantitation (5.97–17.63 pg/band) with good precision (?3.0% coefficient of variation) compared with TLC–densitometry. The proposed indirect HPTLC–MS method is simple yet effective and has tremendous potential in the separation and quantitation of drugs and their metabolites from biological samples, especially for clinical studies.     

Determination of Oxazepam in Pharmaceutical Formulation by HPTLC UV-Densitometric and UV-Derivative Spectrophotometry Methods

Abstract

Methods for determination of oxazepam in pharmaceutical formulation by derivative ultraviolet (UV) spectrophotometry as well as high-performance thin-layer chromatography (HPTLC) UV densitometry were described. For UV-derivative spectrophotometry, some derivatives and wavelengths may be recommended for routine quality control of the drug of interest. On the other hand, HPTLC provided good results, but only when the calibration curve was estimated using nonlinear regression analysis. The HPTLC method was developed with silica F₂₅₄ plates, a mobile phase of benzene/ethanol (5:1, v/v), and densitometric detection at 204 nm receiving R _f  = 0.47. Developed methods were validated and found to be sufficiently precise and reproducible for established conditions.     

Determination of N,N-Dialkyl Dithiocarbamates in Wastewater by Thin-Layer Densitometry

Abstract

The quantitative determination of N,N-dialkyl dithiocarbamates tetramethylthiuram disulfide and tetramethyl-thiuram monosulfide in industrial wastewater was studied by thin-layer densitometry of their copper complexes. It has been demonstrated that a densitometer provides a simple and rapid means to quantitate these compounds. N,N-dialkyl dithiocarbamates have been analyzed in microgram quantities using this method. Coefficients of variation of 2–6 per cent were obtained.     

The synthesis and morphology of grafted semiinterpenetrating polymer networks on the basis of functional prepolymers

Semi-IPNs on the basis of PU prepolymers with carboxylic groups and methacrylic copolymers with tertiary amine groups were synthesized. The influence of the functional group content and of the M_n of polymer components, as well as their ratio, on the morphology and mechanical properties were investigated. The miscibility in the systems was estimated by differential scanning calorimetry (DSC) and by scanning electron microscopy (SEM). The miscibility of components was increased by augmenting the content of the functional groups. By varying the ratio of components, the highest miscibility was found at the isoelectric ratio of functional groups. However, all the investigated IPNs were two-phase systems, due to the segmented structure of PU prepolymers. The lowering of M_n values of the starting components did not improve their compatibility. The mechanical properties of semi-IPNs changed with the increased concentration of functional groups in a way that is typical for ionomers. By physical interactions between functional groups, Young's modulus was most influenced, while the M_n of the polymethacrylic component influenced the tensile strength of IPNs. The compatibility between PU hard segments and the polymethacrylic component found in our experiments led us to the conclusion that the preparation of IPNs of finer morphology would be possible by using PU prepolymers with shorter soft segments. © 1996 John Wiley & Sons, Inc.     

Correlation Between Structure and Transport Properties of Polymeric Membranes for Immunoisolation

Laboratory-made asymmetric polyurethane membranes designed for immunoisolation were investigated. Two types of EK and ES membranes were prepared in different spinning conditions. The membrane structure was characterised by the skin pore radius measurements using differential scanning calorimetry (DSC). Diffusive transport properties of membranes were determined by in vitro method for albumin and creatinine. The scanning electron microscopy (SEM) was applied to study the morphology of membranes. It has been found that the DSC technique is a useful tool for the evaluation of pore radii in the skin layer for PU membranes. Calculated pore radii were in the range from 1.95 to 2.47 nm for the EK and ES types. A correlation between the skin pore radii and the transport properties was not found in this case of investigated membranes. However, the transport properties data can serve for the estimation of selectivity of membranes. Thus, the selectivity of membranes for solutes of various molecular size was estimated from the D _m/D _w ratio of diffusion coefficients for albumin and creatinine. The SEM micrographs reveal the finger-like internal structure of capillary membranes, as well as various skin thickness. This revised version was published online in July 2006 with corrections to the Cover Date.     

Investigation of a Novel Microtiter Plate Support Material and Scanner Quantitation of Immunoassays,Proteins and Phospholipids

Abstract

A recently developed shallow-well microtiter plate, made from a specially formulated polymer that binds proteins, peptides and nucleic acids rapidly and efficiently, has been investigated for use in solid-phase immunoassays as well as for protein and phospholipid assays. The assay uses quantitation of the color intensity of a dye on the solid phase by means of a simple desk-top scanner coupled to a computer which allows the gray-scale density of the color to be easily and accurately measured. Consequently, this approach is independent of the absorption spectrum of the dye used.

These studies demonstrate that the novel technology incorporated into these unique shallow-well microtiter plates has potential in both protein and immunoassays. In the latter case, there are considerable savings in both time and material costs over conventional ELISA methods. The scanning software provided for the solid-phase immunoassay is user configurable for other densitometry assays on different solid matrices and can be read directly into a spreadsheet file for subsequent data manipulation.     

Determination of Parabens and their Degradation Product p-Hydroxy-Benzoic Acid in Pharmaceutical Dosage Forms by Hptlc Densitometry

Abstract

HPTLC densitometry has been developed for the determination of parabens and their degradation product p-hydroxybenzoic acid in pharmaceutical preparations. The method was used for simultaneous determination of parabens in combination:methylparaben - propylparaben, propylparaben - butylparaben and for the determination their stability in dosage forms (suspensions, ointment). The substances were separated on silica gel with fluorescence indicator in system n-pentane - glacial acetic acid (88:12, v/v). Absorbance measurements(detection of reflectance) of separated parabens was carried out “in situ” at 256 nm using single-level calibration (external standard). For the determination of p-hydroxybenzoic acid, a five-level calibration curve (external standard) was used in the concentration range of 20–100 ng/band. The results were evaluated by linear regression analysis. The relative standard deviation values (R.S.D.%) were in range 1.24–1.89 %.     

Novel Optical Measurement Approach for the Quantitation of Liposome Immunomigration Assays

Abstract

In a recent series of papers^1–3 the use of dye-containing liposomes as a component of immunochemical field assay methods for the detection and measurement of environmental contaminants has been demonstrated. These assays produce a colored measurement zone on a plastic-backed nitrocellulose strip, with the amount of color in the zone being proportional to the amount of analyte present in the test sample. The color in this zone has been measured using scanning densitometry of the strips. While giving a quantitative measurement, such a system is not field-portable and cannot differentiate between multiple colors in the measurement zone. The human eye is also limited in the case of mixed colors. In this report, the use of a Colortron^TM 32-band digital color sensor for the quantitation measurement is demonstrated. This instrument is designed for inexpensive color analysis in the desktop publishing industry, but the fact that it can be used as a portable scanning reflectometer means that it has great potential for the spectral analysis of colors produced on a solid surface as analytical signals. As it is a scanning reflectometer rather than a single wavelength instrument, it can discriminate and quantitate a mixture of dyes, and thus has the potential to become part of a field-portable, multianalyte assay system.