In our project, our team aimed to obtain Parkinson's disease (PD)-specific markers NF-L, Aβ42, and α-syn by
genetic engineering methods. Finally, we expect to prepare for the acquisition of specific antibodies and the
construction of early PD diagnosis kits. Throughout out engineering cycle, we designed two major quantitative
experiments requiring categorical measurements:
(1) Western blotting assay: Confirming the best induction condition for protein expression of
pET-28a(+)-NF-L/Aβ42/α-syn recombination plasmids;
(2) Sandwich ELISA assay: Confirming the accuracy and quantifying the yield of recombination proteins purified
by His-tag affinity purification kit;
Western blotting assay was critical to the ‘Build’ phase of our Engineering Cycle, whereas sandwich ELISA
assay was crucial to the ‘Test’ phase. First, the western blotting assay needs a specific antibody for His-tag
and ImageJ software to quantify the expression levels of protein (i.e., the gray value intensity of protein
bands). Second, the sandwich ELISA assay requires a standard curve to quantify the concentrations of target
proteins. Accordingly, our measurement protocols were the following:
(1)Western blotting assay: Obtaining the gray value intensity of target protein bands that could reflect the
protein yield under different induction conditions;
(2)Sandwich ELISA assay: Obtaining three standard curve that could used to quantify the yield of NF-L, Aβ42,
and α-syn purified recombination proteins, respectively;
Measurement Background
Western blotting assay[1], proposed by Towbin et al in 1979, is now widely used for detecting specific
protein. The specificity of antibody-antigen interactions enables target protein recognition in complex
protein mixtures. ImageJ software can obtain the qualitative data on target proteins followed by western
blotting assay.
Measurement Principle
In Western blotting assay, the procedures are briefly described as following. First, the proteins are
separated by SDS-PAGE. After separation, the proteins are transferred from the gel onto a PVDF membrane
through electroblotting. The membrane is then blocked to prevent non-specific binding, followed by incubation
with a primary antibody that specifically binds to the target protein. After washing off excess antibodies, a
secondary antibody, linked to an enzyme (e.g., horseradish peroxidase), is added. This antibody binds to the
primary antibody. After the proteins are detected by specific antibodies, the bands are usually visualized
through chemiluminescence (ECL) dyes. The intensity of these bands corresponds to their optical density.
ImageJ is capable of calculating the pixel intensity values (gray values) of the image, which represent the
strength of the protein bands.
Measurement Protocols
Materials:
(1)PVDF membrane with target proteins
(2)ECL dye solutions
(3)Chemiluminescent detection digital imager
(4)ImageJ software
Procedures:
(1)Selection of ROI (Region of Interest): one of the key steps in ImageJ quantification is accurately
selecting the ROI, or the area containing the band of interest (Figure 1). ImageJ measures the integrated
density of the pixels in this region, summing the pixel intensity values. The integrated density is directly
proportional to the amount of protein in the band;
Figure 1 Calculation methods of the intensity of ROI.
(2)Calculation of gray values integrated density: ImageJ software uses grayscale values to represent pixel
brightness, typically ranging from 0 to 255, where 0 is black (high density) and 255 is white (low density).
ImageJ software calculates the gray value integrated density by summing all the pixel values within the
selected ROI, providing a quantitative measure of protein content (Figure 2).
Figure 2 Raw data of the gray value integrated intensity of target proteins.
(3)Plot according the gray value integrated density of target proteins under different induction time and
temperature (Figure 3).
Figure 3 The relative gray value intensity of target proteins expression
From the images of western blotting assay, we found that the His antibody specificity is not particularly
high, suggesting that we might need to optimize the relative concentrations of the primary and secondary
antibodies, but we can still clearly see the bands of target proteins. Then, we used ImageJ software to
calculate the relative gray value intensity of target proteins. According to the gray value intensity, except
for α-syn protein, we chose 25°C and 37°C after 6 hours of induction for large-scale purification. For α-syn
protein, we chose 25°C after 1 hours of induction and 37°C after 6 hours of induction for large-scale
purification, respectively. At 25℃, α-syn protein might form insoluble inclusion bodies or precipitate in
E.coli, resulting the reduction of soluble protein. It might cause a seemingly decreased expression level of
α-syn protein under 25℃.
Measurement Background
Sandwich ELISA assay[2], a highly versatile and widely adopted technique in antibody-antigen detection, is a
crucial tool for measuring target protein concentration. This assay relies on building a standard curve for
quantifying the expression level of target protein in unknown samples.
Measurement Principles
ELISA (Enzyme-Linked Immunosorbent Assay) is a widely used method for detecting and quantifying proteins. The
mechanism involves the binding of specific antibody to its target protein, followed by a secondary
enzyme-linked antibody that catalyzes a colorimetric reaction. In order to detect the concentrations of target
protein in samples, we need firstly to construct a standard curve. Standard known concentrations of substance
are prepared and assayed in parallel with the samples. Then, the biotin-labeled antibody is incubated with
samples simultaneously. After washing, add avidin-labeled HRP. After incubation and washing, the unbound
enzyme binding is removed, and then substrates are added to act with the enzyme binding at the same time. The
optical density (OD) is measured at a specific wavelength (i.e., 450 nm) after adding the substrate for the
enzyme. A plot of OD values against the corresponding concentrations generates the standard curve. Fitting
this curve, typically using linear regression methods, allows for the determination of unknown sample
concentrations based on their OD values.
Measurement Protocols
Materials:
(1)Protein samples
(2)Standard NF-L protein solution (125 ng/mL)
(3)Standard Aβ-42 protein solution (80 pg /mL)
(4)Standard α-syn protein solution (325 ng/mL)
(5)Sample diluent
(6)Washing buffer
(7)Clear, flat-bottom 96-well microplate coated specific antibodies
(8)Substrate solution
(9)End solution
(10)96-well microplate absorbance reader
Procedures:
(1)Prepare standard target protein solution (take NF-L as an example):
Figure 4 Preparation of NF-L standard solutions.
The following condition was used to prepare the five samples of standard curve sample (Figure 4). We diluted
125 ng/mL NF-L protein solution via sample diluent to obtain five standard samples with NF-L concentrations of
125, 62.5, 31.25, 15.625, and 7.8125 ng/mL, respectively. For Aβ-42 protein, we obtained five gradient samples
of 80, 40, 20, 10, and 5 pg/mL. For α-syn protein, we obtained five gradient samples of 325, 162.5, 81.25,
40.625, and 20.3125 ng/mL;
(2)Add 100 µL of each diluted sample and standard samples to 96-well microplate;
(3)Cover and incubate for 1-2 hours at room temperature;
(4)Wash the wells 3-5 times with washing buffer;
(5)Prepare a dilution of the detection antibody in sample diluent;
(6)Add 100 µL of the diluted detection antibody to each well;
(7)Incubate for 1-2 hours at room temperature;
(8)Wash the wells 3-5 times with washing buffer;
(9)Add 100 µL of the substrate solution to each well. This substrate solution will react with HRP enzyme linked
to the detection antibody;
(10)Allow the color to develop for 30 minutes at room temperature in the dark;
(11)Add 50 µL of stop solution to each well to terminate the enzymatic reaction (Figure 5);
Figure 5 ELISA assay standard sample using a 96-well microplate
(12)Measure the absorbance of each well at 450 nm wavelength using microplate reader;
(13)Plot a standard curve using Excel to show the known concentrations of targe protein standard and their
corresponding absorbance values (Figure 6);
Figure 6 The standard curves were used for quantifying NF-L/Aβ-42/α-syn concentrations in purified protein.
(14)The quantification process employed the trendline equation derived from the standard curve. This equation
directly correlates the absorbance values obtained from our samples and the corresponding protein
concentrations. We determined the protein concentrations for each condition examined in our experiment using
this established relationship. Using the equation of the standard curve in Figure 6, the total protein
concentrations were calculated:
NF-L concentration (ng/mL) = [Absorbance (450 nm) + 0.0603] / 0.0133
Aβ-42 concentration (pg/mL) = [Absorbance (450 nm) + 0.0863] / 0.0204
α-syn concentration (ng/mL) = [Absorbance (450 nm) + 0.0898] / 0.0047
Measurement Discussions
Before the Sandwich ELISA assay, we used the standard in the kit for standardized reference. The standard
already had the corresponding protein concentration value. First, the standard was diluted in a gradient, and
then the absorbance of the diluted standard at 450 nm was measured ( Figure 5 ). After the measurement was
completed, a linear regression equation between absorbance and protein concentration was plotted as the
standard curve we would use next[3]. The results showed that there was a strong positive linear correlation
between the absorbance values of the three proteins and the protein concentration ( R2 = 0.99 ), which
verified the reliability of predicting the total protein concentration from the measured absorbance ( Figure 6
). Therefore, we can quantify the total protein concentration of NF-L / Aβ-42 / α-syn after BL21 induced
expression, and verify the molecular integrity of the pET28a vector system after transformation.
In order to obtain a calibrated regression equation, we also set up a control for the standard : a standard
diluent with a concentration of 0 ng / mL or 0 pg / mL, the X-axis is the concentration of the protein, and
the Y-axis is the absorbance of the corresponding protein at 450 nm. The explanatory variables and response
variables were measured and presented in the form of a least squares regression line with strong positive
linear correlation. We strongly recommend that future iGEM teams use such control / calibration measures to
obtain the correct data. This measurement technique may be helpful to teams that lack an appropriate
standardized reference when quantifying total protein concentration. However, it may be difficult to obtain
the relative abundance or molar ratio between multiple proteins using this method, which requires further
calculations.
