Quantification of Soluble or Insoluble Fractions ofLeishmaniaParasite Proteins in Microvolume Applications: A Simplification to Standard Lowry Assay

Abstract

Protein quantification is often an essential step in any research field that involves proteins. Although the standard Lowry assay and its modifications are most abundantly used in protein quantification, the existing methods are rigid or often demonstrate nonlinearity between protein concentration and color intensity. A method for fast and accurate qualitative and/or quantitative determination of total soluble/insoluble proteins or micro-well plate immobilized proteins isolated from Leishmania parasites in microvolumes was described in the current study. Improvements in cost-effective techniques are necessary to increase the research outputs in resource-limited settings. This method is a modification to the established Lowry assay for protein quantification. Concentrations of unknown samples were calculated using a standard curve prepared using a standard series of bovine serum albumin (BSA). The optimized reagents were 2 N NaOH (sodium hydroxide), 2% Na2CO3 (sodium carbonate), 1% CuSO4 (copper sulfate), 2% KNaC4H4O6 (potassium sodium tartrate), and 2 N Folin and Ciocalteu’s phenol. This modified protein assay was sensitive for quantifying Leishmania proteins in a total crude extract or in a soluble fraction within the approximate range of 10–500 μg/ml (1–50 μg/assay) and showed a linearity between color intensity and concentration of the protein. This is an easier, fast, and accurate method for quantifying proteins with microvolumes in a cost-effective manner for routine use in research laboratories in resource-limited settings.

First Serological Study Revealing High Humoral Response and Evidence for Antigenic Heterogeneity in Leishmania donovani Induced CL in Sri Lanka

“…Suspension was freeze-thawed for three times (freezed in liquid nitrogen and thawed at room temperature). Crude Ag was quantified using a modified Lowry assay [ 25 ]. It was aliquoted and stored at -20°C.…”

Physico-chemical and gel properties of heat-induced pasteurized liquid egg white gel: effect of alkyl chain length of alcohol

“…The first scan was of native protein and the second scan was of heat-treated protein. The data were normalized with respect to protein concentration, determined using Lowry's method [45,46] with BSA as a standard protein. Peak temperature (T m ) and enthalpy (ΔH) were analyzed using the STAR e Thermal Analysis software 14.00.…”

Prevalidation of an ELISA for Detection of a New Clinical Entity: Leishmania donovani-Induced Cutaneous Leishmaniasis

“…According to the modified Lowry assay method, the yield of the crude Ag extract was 15 mg for 1 g of the parasite cell pellet [ 36 ]. Selected amounts of crude Ag (0.5 μ g, 1.5 μ g, and 2.5 μ g) were initially used for coating the microwell plate, and the outcome of ELISA was checked.…”

“…Extracted crude Ag was quantified using a modified Lowry assay which was developed and validated within home laboratory settings [ 36 ]. Briefly, BSA was used as the standards (100–500 μ g/ μ l).…”