This method demonstrates the calibration of quantifying protein amount in liquid sample with single-molecule process for counting. Specifically, it can illustrate how protein populations can be fluorescently labeled at each molecule level. Previous methods performed bulk measurements to analyze molar ratios of fluorescent and protein molecules, but they cannot reveal how heterogeneously protein populations are actually labeled.
Our methods can do it directly at the single-molecule level. Our method can be extended towards ultrasensitive and advanced molecular concentration assays, which will lead to future diagnostic methods, allowing efficient findings of pathogenic molecules in specimens. Our method can be applied to single protein species analysis using fluorescent antibodies, as well as multiple species analysis using non-specific labels for all the proteins separated by electrophoresis or chromatography.
The most critical point of the technique is obtaining data reproducibility. I recommend keeping the same experimental condition as much as possible when measuring multiple samples. Visual demonstration of this technique will provide how to make each experimental step stable and reproducible.
It will also show some uncommon step in biochemistry protocols, such as the spin-coating and washing of microscope coverslips. To begin this procedure, prepare the lysing buffer and 1%Tween-20 as outlined in the text protocol. Use sodium hydroxide to adjust the pH to 12.
Mix 100 microliters of lysing buffer with one microliter of one molar DDT and four microliters of 50%CHAPS. Add one microliter of the prepared cell culture and homogenize the solution by slow pipetting to avoid bubbles. Incubate in the dark at room temperature for five minutes with gentle agitation.
Then, rehomogenize the solution by pipetting it up and down slowly. Add one microgram of Cy3 NHS ester dye and homogenize the solution by slowing pipetting to avoid bubbles. Incubate in the dark at room temperature for 10 minutes with gentle agitation.
Repeat this process, adding the same amount of dye and incubating the sample one time. Add 100 microliters of 0.8 molar HEPES-sodium hydroxide to adjust the pH of the solution to 7.2 and to quench the reaction. Slowly pipette up and down to homogenize the solution.
Next, add 20 microliters of Biotin-2-amine and five microliters of EDC. Homogenize the solution by pipetting up and down slowly. Incubate in the dark at room temperature for one hour with gentle agitation.
After this, use an ultrafiltration column with a 10 kiloDalton cutoff to remove any unreacted labeling reagents and concentrate the sample. After performing standard SDS-PAGE for the protein sample and a molecular ladder, image the gel to identify the location of the protein bands. Use a sharp blade to cut out the gel portions that include protein fractions of interest based on the bands'locations.
First, set out coverslips that are 22 millimeters by 22 millimeters and are 0.15 millimeters thick. Use a plasma cleaner to expose the coverslips to air plasma for one minute to clean and activate their surfaces. Next, use a spin coater to spin coat the coverslips with 200 microliters of avadin buffer for five seconds at 500RPM, followed by 30 seconds at 1000RPM.
Incubate the coverslips at room temperature for 15 minutes to let them air dry. To prepare coverslips for the protein sample, use a protein sample that has been diluted. Place 100 microliters of the diluted sample onto the center of the avadin-coated coverslips.
Incubate in the dark at room temperature for 15 minutes. To prepare the positive control, place 100 microliters of purified fluorescent biotin in five micromolar HEPES-sodium hydroxide at pH 7.2 in the center of an avadin-coated coverslip. Incubate in the dark at room temperature for 15 minutes.
To prepare the negative control, spin coat plasma coverslips with 200 microliters of five HEPES-sodium hydroxide with two milligrams per milliliter of BSA without avadin. Add 100 microliters to purified fluorescent biotin in five micromolar HEPES-sodium hydroxide at pH 7.2. Incubate in the dark for 15 minutes at room temperature.
After this, rinse each coverslip with 200 microliters of distilled water by pipetting at the edges, making sure to not touch the middle of the coverslip with the pipette tip. Repeat this wash three times. Then, expose an equal number of new coverslips to air plasma for one minute.
Place a cleaned coverslip on top of each sample-bound coverslip to avoid drying. Start up a wide-field or evanescent field fluorescence microscope. Set a coverslip onto the microscope and find the focus.
Then, perform a tile scan to obtain at least 100 images. In this study, the labeling homogeneity of each labeled protein species in cells is quantified after separation with SDS-PAGE. The raw image data for different molecular weight fractions of proteins from HeLa cell lysate, as well as the positive and negative controls, are seen here.
While both the protein sample and the positive control exhibit between 100 and 500 spots per image, the negative control exhibits very few to none. This shows that the process sufficiently inhibits non-specific binding of dyes to the coverslip surface. Spot intensity histograms for the protein samples and the positive control present multiple peaks, which represent the stochastic binding of dyes to primary amines in proteins and avadin tetramer structures respectively.
All spots showed blinking and stepwise photobleaching with continuous laser excitation, highlighting observation at the single molecule level. The labeling occupancy, or LO, is then calculated from the ratio of the number of spots in every protein sample to that in the positive control. The LO measured from the HeLa cell lysate sample ranges from 50%for the smaller molecular weight fraction, to 90%for the higher molecular weight fraction.
The LO for the whole proteome sample without separation is seen to be 72%It is critical to use freshly made reagents and to avoid any dust, especially during the preparation of the coverslips. Following this procedure, single molecule counting analysis in a certain volume can be performed to quantify each protein's concentrations. For example, analysis on any raw product, or gel and capillary electrophoresis products, can be performed.
This technique paves the way for utilizing single molecule fluorescence imaging to molecular medicine, organic chemistry, and omics analysis. By bridging the concept between concentration and numbers. Concentrated sodium hydroxide is corrosive, and adequate protection should be worn when handling it.
Also, high power laser radiation can cause eye damage, so protective goggles may be worn.
