On the use of ratio standard curves to accurately quantitate relative changes in protein levels by Western blot

Abstract

Relative changes in the expression of a specific protein are commonly measured on western blots by forming the ratio of the densitometric values of bands containing the protein between control and experimental samples. It is generally assumed that this analysis provides an accurate determination of relative changes in a specific protein expression if there is a linear relation between increasing amounts of that protein, as represented by bands on a western blot or a gel, and the densitometric measurements of these bands. In this study, we provide direct evidence that this assumption is invalid because even in the presence of a linear relation, densitometric ratios differ substantially from know actual ratios of protein amounts. We present also the mathematical basis for this bias, and demonstrate that it can be circumvented by using a "ratio standard curve". Alternatively, the use of purified protein standards to plot a standard densitometry curve can solve this problem, but a survey of the literature shows that most investigators who use western blots to quantify relative changes in protein expression samples do not have standards for the specific proteins that they are investigating.To examine whether densitometric ratios accurately reflect ratios between protein amounts, we performed experiments to silence lamin C (a nuclear envelope protein) in U-373 MG glioblastoma cells. These cells were grown as described earlier [1], and were transfected with a lamin C specific siRNA (5′-CUGGACUUCCAGAAGAdTdT-3′ and 5′-UGUUCUUCUGGAAGUCCAGdTdT-3′ for the sense and anti-sense sequences, respectively) using siLentFect lipid reagent (Bio-Rad) according to the manufacturer's instructions. Control cells were treated with transfection reagent and vehicle. One and two days after transfection, the cells were processed for SDS-PAGE as described earlier [1]. Protein concentrations were measured with the bicinchoninic acid assay [2]. Gels were loaded with incremental amounts (4, 8, 20, 40, and 80 g) of total cellular proteins from control samples, as well as with 80 g of total cellular protein for experimental samples. Each loading was performed in duplicate, and all the loadings were applied to the same gel to insure identical treatment during the western blotting procedure, which was performed following standard methods [3]. In addition, to avoid band distortion, sample loadings were brought to the same final volume with sample buffer. After blocking in PBS plus 5% dry milk, the blots were incubated with a rabbit anti-lamin antibody (Chemicon, Temecula, CA) diluted at 1:100 in blocking solution, and then with goat anti-rabbit IgGs conjugated to peroxidase (KPL, Gaithersburg, MD) diluted at 1:1000 in blocking solution. After incubation of the blots Address correspondance to: Dr.

Turnover of glycosomes during life-cycle differentiation ofTrypanosoma brucei

“…ALD levels decreased progressively, whereas the cells underwent a considerable depletion of PYK between 60 min and 6 h. These results have to be interpreted with care because the method used only registers the tendency of proteins to increase and decrease and does not allow a precise quantification. 40 Nonetheless, the mechanisms by which these two glycolytic proteins are degraded might be different, because ALD is glycosomal and, as shown above, is degraded with the organelle by pexophagy while PYK is cytosolic and would probably follow another degradation route. It would be interesting to compare the degradation of PYK with other cytosolic enzymes, also some not involved with glycolysis.…”

“…LS, long-slender cells; I1, mixture of long-slender and intermediary trypanosomes; I2, mixture of intermediary and short-stumpy trypanosomes; SS, short-stumpy cells; I3, short-stumpy cells differentiating into procyclics; PRO, procyclics; N, nucleus; K, kinetoplast. Immunofluorescence studies were carried out on bloodstream-form cells taken from the mice each day from day 3 (~90% long-slender) to day 6 (~ 90% short-stumpy) and on cultured cells taken at 0, 5,10,20,30,40,50,60,90, 120 min and 24 h after incubating the short-stumpy trypanosomes at conditions allowing their differentiation to procyclics. Preparations for immunofluorescence studies were incubated with both 'an anti-aldolase (ALD) antiserum and an anti-p67 monoclonal antibody to stain specifically glycosomes and lysosome, respectively, in order to obtain indications as to whether glycosomes are degraded in the lysosome.…”

Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

“…Proteins and LPS were both visualized by silver staining. The amounts of protein and LPS in each lane were determined by densitometry using ChemiImager (AlphaInnotech) and quantified accurately by using ratio standard curves (48). The relative amount of LPS to protein (in arbitrary units) at each concentration of LPS was plotted.…”

σ-1 Receptor at the Mitochondrial-Associated Endoplasmic Reticulum Membrane Is Responsible for Mitochondrial Metabolic Regulation

“…The band intensity was determined by using Image Quant 5.2 (Phosphorimager; GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). The densitometric calculation in each figure reflects the ratio of -1 or VDAC2 and ␤-actin following an established procedure (Pitre et al, 2007).…”