High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.


Introduction
Alongside with the exciting advancements in genomic research in the recent years, biomedical research field also witnesses the significant advancement in proteomic research. With increasing accumulation of biological data at both genomic and proteomic levels, using bioinformatic tools to analyze these data has become the focus of biomedical research in the perceivable future. Consequently, the success of bioinformatical research raises demand for more and better quality of data from the biological and biomedical research community, a task can only be achieved through technique advancement at genomic and proteomic levels.
Mass spectrometry (MS) and immunoblot analysis are two prevailing techniques of protein analysis presently. MS has dominated the proteomic research in the recent years to enable analysis of thousands of individual proteins simultaneously. The immunoblot-based techniques, including western blot and dot blot, on the other hand, have also played a significant role in the protein research even since its invention 1,2,3,4,5 . Enzyme linked immunosorbent assay (ELISA) 5,6,7 and reverse phase protein microarray (RPPM) 8,9 can be considered the high throughput format of immunoblot analysis. However, all these immunoassay methods, except ELISA, measure the relative expression level of a specific protein. The relative nature of these methods will become a real problem for population studies, as the analysis must be done at the same time, preventing any efforts to increase the pool size through multiple analyses. Furthermore, the results derived from these studies are only semi-quantitative, thus complicating any bioinformatics efforts in the data analysis. Meanwhile, although ELISA is well suited for high throughput absolute analysis of protein samples, this technique seems to meet challenges in complex environments such as cells or tissues because of its low binding capacity and multiplexing 10 .
We have developed an immunoblot method suitable for population studies with the key characteristics of being convenient, high throughput, quantitative, and suitable for absolute determination of protein content, and named this technique quantitative dot blot analysis (QDB) 11 . In this study, we present a detailed protocol for QDB analysis and demonstrate the method by determining the absolute protein content of a specific protein, CAPG, in three different mouse tissues including kidney, spleen, and prostate. We think that this detailed protocol well illustrates the feasibility and convenience of this method, and provide guidance on how to avoid the potential pitfalls in the practice of this method.

Representative Results
The determination of the absolute amount of any protein including the CAPG protein in mouse tissue requires both a specific antibody and a purified protein as standard. The linear range of the analyses of both the protein standard and the lysates also needs to be established prior to any large-scale analysis. The linear range of the antibody is highly dependent on the antibody per se, and the appropriate dilution range of a specific antibody need to be verified by each individual user.
We first determined the specificity of the CAPG antibody in mouse kidney, spleen and prostate tissues using western blot analysis with the negative control (IgG free BSA) and positive control (commercially available CAPG protein) ( Figure 1A). The anti-CAPG antibody was specific against lysates from mouse spleen, heart, muscle, and prostate (one band detected). Non-specific bands were observed in lysates from kidney and liver. However, in kidney lysate, the non-specific bands were significantly weaker than the specific band, which suggests the antibody is suitable for kidney tissue analysis. Next, the linear range of the protein standard and the amount of lysates for analyses were determined by two side by side dose curves. A commercially available CAPG protein was serially diluted as indicated in Table 1 based on our past experiences, and the tissue lysates of the mouse kidney, spleen and prostate were also diluted based on the amount of total protein in the lysates determined by a BCA total protein determination kit. Two dose studies were performed side by side and plotted together in Figure 1B. The appropriate amount of lysates of mouse kidney, spleen and prostate were chosen based on the QDB signals measured by the microplate reader in arbitrary unit. The original reading for this experiment is shown in Table 3.
In the last step, the serially diluted CAPG protein standard and the lysates from mouse kidney, spleen and prostate were loaded onto the QDB plate. In this case, we chose to load 0.3 µg/sample for lysates from mouse kidney and spleen and 1 µg/sample for lysates from mouse prostates analysis, as at these levels, the QDB reading was at least 20-fold over the background while well within the linear range of the analysis based on the dose curve of both the lysates and the protein standard. The plate was subjected to the described QDB protocol before the plate was quantified directly by the microplate reader. Using the serially diluted purified CAPG protein, we could establish a dose-response curve, the equation, and R2 by simple regression analysis using available software (e.g. Microsoft office excel). The QDB signals from lysates of mouse kidneys, spleens and prostates were converted to the absolute amount of CAPG protein in these lysates using the established equation, and the results were corrected by the total protein amount, in this case, 0.3 µg for lysates from mouse spleens and kidneys, and 1 µg for lysates from mouse prostates, for the final concentration of CAPG protein in these tissues as pg/µg. The results are shown in Figure 1C with the original reading shown in Table 4.
Compared to ELISA, QDB analysis requires minimum efforts to be developed in a regular lab. First, the QDB plate is a based on a nitrocellulose membrane to eliminate the coating step in ELISA. Second, QDB analysis only requires one instead of two specific antibodies in a sandwich ELISA. Third, the high binding capacity of nitrocellulose membrane as compared to the ELISA plate surface also allows the membrane to withstand the stringent washing steps typically used in the immunoblot analysis to reduce the background interference. This feature is very useful in analyzing complicated lysates prepared from cells and tissues. In contrast, due to the relatively low binding capacity of ELISA plates, the reduction of the background when analyzing complicated cellular and tissue lysates becomes a real challenge in the developmental process.
QDB analysis can be adopted easily in any lab with the access of a specific antibody. However, it is important to mention that the specificity of the antibody is a relative term, limited by factors including the species, the tissue types and cell types. As shown in Figure 1A, CAPG antibody is specific when analyzing lysate from mouse kidney, spleen, and prostate, and yet becomes non-specific when analyzing mouse liver. In fact, we routinely found one antibody to be specific for one tissue type, but not always specific to other tissue type from the same species. Thus, prior western blot analysis is necessary to ensure the specificity of the QDB analysis. In fact, the relative specificity of the antibody may be the cause of false results often associated with ELISA analysis, as no matter how much effort the manufacturing company may have spent to ensure the specificity of the assay, they cannot exhaust all the possible types of samples the users may choose to analyze using their ELISA products.
As with all immunoassays, a potential problem with the QDB analysis method is the lack of consistency of the quality of the commercial antibody. Even if the apparent quality of an antibody from the same company (same catalog number, etc.) is used, there could be large differences from one purchase to another due to batch-to-batch variability. Therefore, unless full confidence is attained in the quality of the antibody available, recharacterizing the antibody upon every purchase is important.
In summary, we provide here a detailed protocol and an example when using the QDB analysis method to achieve absolute quantitative determination of a specific protein at the tissue level. We show that the QDB analysis method is a convenient tool for anyone who is interested in high throughput, quantitative immunoblot analysis. Its ability to achieve absolute determination of the content of a specific protein at a cellular and tissue level also distinguish this technique from traditional immunoblot techniques. This feature allows for the combination and comparison of results from multiple analyses, a step necessary especially in larger population studies, and the realization of relevant association studies at the protein level in the near future.

Disclosures
The authors Yunyun Zhang, Wenfeng Zhang and Jiandi Zhang are employees of Zestern Biotechniques that produces QDB plates used in this Article. Wenfeng Zhang and Yunyun Zhang declare conflict of interests, and Jiandi Zhang has filed patent applications. The others claim no competing interests.