![]() The protocol was applied to the quantification of the periplasmic production of a recombinant nanobody that neutralizes specific toxins in the scorpion venom.Įxperiments were conducted with Escherichia coli K12/WK6 harboring pHEN6 plasmid (derived from pBR322) encoding the chimeric format of the bispecific nanobody VHH10‐VHHF12 (called CH10‐12), retrieved from the combinatorial libraries. The molecular weight marker is then used as a sole protein standard for protein quantification and molecular weight estimation. The unknown proteins are quantified with commercially available protein standards: bovine serum albumin (BSA), carbonic anhydrase (CA), and ovalbumin (OV), and molecular weight markers of known concentration. The protocol describes the quantification of protein in the bands of SDS‐PAGE gels. In the present work, a new protocol of image analysis for electrophoresis gels is reported. The volume of the peak is not commonly considered (Vincent et al., 1997) since the correlations obtained often yield inaccurate protein load estimation (Gassmann et al., 2009). The peak area is the signal used in most densitometry analysis (Gassmann et al., 2009 Gorr & Vogel, 2015 Rehbein & Schwalbe, 2015), and the peak maximum intensity has been utilized in the quantification of proteins in Western blots imaged by fluorescence (Gürtler et al., 2013 Holzmüller & Kulozik, 2016). Image analysis by densitometry is made using the profiles of the lanes and calibrated to a known standard (Cromey, 2010 Syrový & Hodný, 1991). ( 2013), in which the ultraviolet fluorescence of the protein of interest was used as the quantifying parameter of the unknown samples. Stain‐Free TM technology by Bio‐rad (USA) has been applied by Holzmüller and Kulozik ( 2016) and Gürtler et al. The use of external protein standards of known concentration is commonly used for the quantification of the electrophoresis‐based separated samples (Holzmüller & Kulozik, 2016 Rehbein & Schwalbe, 2015 Vincent, Cunningham, Stephens, Halayko, & Fisher, 1997). The semiquantification of protein load in the gel is made through a nonstandardized methodology, using densitometry in SDS‐PAGE and Western blot assays (Gassmann, Grenacher, Rohde, & Vogel, 2009). SDS‐PAGE is used to check and characterize the purified recombinant proteins, and colorimetric and ultraviolet absorption methods are used to quantify them (Bradford, 1976 Stoscheck, 1990). The identification and quantification of these nanobodies are of the utmost importance during the purification steps. The nanobodies are normally obtained after purification of periplasmic proteins using IMAC (Pardon et al., 2014). The periplasmic production of these nanobodies has been studied in shake flasks induced by synthetic IPTG under the control of the lac promoter (Laustsen et al., 2016). Lately, nanobodies, small fragments of camelid antibodies, have been proven to neutralize scorpion toxins (Alirahimi et al., 2018 Hmila et al., 2008). The molecular weight marker was used as a sole protein standard for protein quantification in SDS‐PAGE gel images.Įscherichia coli is a well‐known microorganism used as a workhorse in the production of recombinant therapeutic molecules, such as antibodies and antibody fragments (Graumann & Premstaller, 2006). The production of the nanobody CH10‐12 was obtained through a fed‐batch strategy and quantified using the band of 50 kDa in the marker as reference for 750 ng of recombinant protein. No brightness and contrast adjustment was applied. Protein load and peak area were linearly correlated, and optimal image processing was then performed by background subtraction using the rolling ball algorithm with radius size 250 pixels. Images of the SDS‐PAGE gels were analyzed using ImageJ, and the lane profiles were obtained in grayscale and uncalibrated optical density. Periplasmic proteins extracted by osmotic shock were purified by immobilized metal affinity chromatography (IMAC). Escherichia coli WK6/pHEN6 encoding the bispecific nanobody CH10‐12 engineered by the Pasteur Institute of Tunisia was cultured in a bioreactor and induced with isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) at 28☌ for 12 hr. This study presents a protocol of image analysis for electrophoresis gels that allows the quantification of unknown proteins using the molecular weight markers as protein standards. In literature, few studies have been reported using image analysis for the quantification of protein in SDS‐PAGE: that is, imaged with Stain‐Free™ technology. The protein purity is generally checked using SDS‐PAGE, where densitometry could be used to quantify the protein bands. ![]()
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