This work is supported by Taishan Scholars Construction Engineering (G.T.), The Shandong Excellent Young Scientist Award (ZR2016JL026 to G. T.), National Natural Science Foundation of China (31771284, 3167070448, and 81641108), Shandong provincial natural science foundation (ZR2016JL026, 2017GSF18103), Shandong provincial science and technology Plan(J14LE01 and J15LK03), Yantai science and technology plan(2015ZH083) and Binzhou Medical University Scientific Research Funds (BY2013KYQD17, BY2013KYQD18), the Swedish Research Council grant 621-2011-4423 and 2015-4870. This work is also sponsored by "Yantai Double Hundred Talent Plan" and "Yantai Hi-Tech Zone Blue Ocean Talent Plan".

All animal procedures were carried out in line with Binzhou Medical University Animal Use Directive and approved by the ethical review board of Binzhou Medical University.
1. Sample Preparation
<ol>
	<li>Take 50 mg mouse tissue into a 1.5 mL Tube. Add 200 &#181;L lysis buffer (50 mM Hepes, pH 7.4, 137 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1 mM MgCl2, 10 mM Na2P2O7, 1% Triton X-100, 10% glycerol), supplemented with protease and phosphatase inhibitors (100 mM NaF, 0.1 mM phenylmethylsulfonyl fluoride, 5 &#181;g/mL pepstatin, 10 &#181;g/mL leupeptin, 5 &#181;g/mL aprotinin).</li>
	<li>Homogenize the tissue with a homogenizer for 1 min on ice.</li>
	<li>Centrifuge for 10 min at 8 000 x g at 4 &#176;C.</li>
	<li>Collect the supernatant into new tubes (1.5 mL) and take out 1&#160;&#181;L for protein concentration assay with BCA kit. 
	Note: All the commonly used lysis buffers for western blot analysis and Dot blot analysis can be used for QDB analysis.</li>
</ol>
2. Determining the Specificity of Antibody
<ol>
	<li>Prepare sample lysates (20 &#181;g) in section 1.4, IgG free BSA (20 &#181;g), standard protein (600 pg), for western blot analysis. 
	Note: IgG free BSA is used as a negative control and the standard protein is used as a positive control. The specificity of the antibody is demonstrated by showing one band of right size using the western blot analysis.</li>
</ol>
3. Defining the Linear Range of the QDB Analysis
<ol>
	<li>Dissolve the standard protein with ddH2O. Dilute the protein to a concentration of 1,200 pg/&#181;L.</li>
	<li>Dilute the concentrated protein from the prepared samples in section 1.4 to 4 &#181;g/&#181;L.</li>
	<li>Dissolve the BSA with ddH2O. Dilute the protein to a concentration of 1,200 pg/&#181;L and 4 &#181;g/&#181;L.</li>
	<li>A series of dilution of standard protein and prepared samples were prepared with the guidance of Table 1 and Table 2.</li>
	<li>Mix 10 &#181;L dilution standard protein or dilution prepared samples and 10 &#181;L 2x loading buffer (120mM Tris-HCl pH 6.8, 20% Glycerol, 4% SDS, 0.2% Bromphenol Blue, 200 mM DTT) together.</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>S1 
			(0pg/&#181;l)</td>
			<td>S2 (33.3pg/&#181;l)</td>
			<td>S3 
			(100pg/&#181;l)</td>
			<td>S3 
			(300pg/&#181;l)</td>
			<td>S5 
			(600pg/&#181;l)</td>
			<td>S6 
			(1200pg/&#181;l)</td>
		</tr>
		<tr>
			<td>standard protein(1200pg/&#181;l)</td>
			<td>0</td>
			<td>1.39</td>
			<td>4.17</td>
			<td>12.5</td>
			<td>25</td>
			<td>50</td>
		</tr>
		<tr>
			<td>IgG free BSA 
			(4pg/&#181;l)</td>
			<td>50</td>
			<td>48.61</td>
			<td>45.83</td>
			<td>37.5</td>
			<td>25</td>
			<td>0</td>
		</tr>
		<tr>
			<td>total</td>
			<td>50</td>
			<td>50</td>
			<td>50</td>
			<td>50</td>
			<td>50</td>
			<td>50</td>
		</tr>
	</tbody>
</table>
Table 1. Dilution Scheme for standard protein curve.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>X1 
			(0&#181;g/&#181;l)</td>
			<td>X2 
			(0.25&#181;g/&#181;l)</td>
			<td>X3 
			(0.5&#181;g/&#181;l)</td>
			<td>X4 
			(1&#181;g/&#181;l)</td>
			<td>X5 
			&#160;(2&#181;g/&#181;l)</td>
			<td>X6 
			&#160;(4 &#181;g/&#181;l)</td>
		</tr>
		<tr>
			<td>Sample(4&#181;g/&#181;l)</td>
			<td>0</td>
			<td>3.125</td>
			<td>6.25</td>
			<td>12.5</td>
			<td>25</td>
			<td>50</td>
		</tr>
		<tr>
			<td>IgG free BSA(4&#181;g/&#181;l)</td>
			<td>50</td>
			<td>46.875</td>
			<td>43.75</td>
			<td>37.5</td>
			<td>25</td>
			<td>0</td>
		</tr>
		<tr>
			<td>total</td>
			<td>50</td>
			<td>50</td>
			<td>50</td>
			<td>50</td>
			<td>50</td>
			<td>50</td>
		</tr>
	</tbody>
</table>
Table 2. Dilution Scheme for prepared samples
<ol start="6">
	<li>Heat the mixtures at 85 &#176;C for 5 min.</li>
</ol>
4. Process of QDB Analysis
<ol>
	<li>Sample application
	<ol>
		<li>Support the QDB plate to avoid the bottom of the plate touching the surface of the table. For instance, use an empty pipette tip box as support.</li>
		<li>Load up to 2 &#181;L of the sample to the center of the membrane at the bottom of the individual unit of the QDB plate.</li>
	</ol>
	</li>
	<li>Drying the plate
	<ol>
		<li>Leave the loaded QDB plate for 1 h at room temperate (RT) or as an alternative leave the loaded plate at 37 &#176;C for 15 min in a well-ventilated space.</li>
	</ol>
	</li>
	<li>Blocking the plate
	<ol>
		<li>Dip the QDB plate in the transfer buffer (0.039 M Glycine, 0.048 M Tris, 0.37% SDS, 20% methyl alcohol) and gently shake the plate for 10 s.</li>
		<li>Rinse the QDB plate gently with TBST (Tris-buffered saline, 0.1% Tween 20) three times, and then wash the plate for 5 min in TBST under constant shaking.</li>
		<li>Block the QDB plate with blocking buffer (5% nonfat milk in TBST (100 &#181;L/well) for 1 h under constant shaking.</li>
	</ol>
	</li>
	<li>Primary antibody incubation
	<ol>
		<li>Dilute the primary antibody in the blocking buffer at a chosen concentration (from 1: 500 to 1:5 000), and add 100 &#181;L to each individual well of an ordinary 96 well plate. Insert the QDB plate into the 96 well plate and incubate the combined plates either for 2 h at RT or overnight at 4 &#176;C under constant shaking.</li>
		<li>Alternatively, place the QDB plate inside a box, and fill the box with blocking buffer 2 to 3 mm above the membrane portion of the plate if the same antibody is used for the whole plate. Add the primary antibody at the chosen concentration, and incubate the plate either for 2 h at RT or overnight at 4 &#176;C by under constant shaking.</li>
		<li>Rinse the plate gently with TBST for three times before it is washed with TBST for three times, each time for 5 min under constant shaking.</li>
	</ol>
	</li>
	<li>Secondary Antibody incubation
	<ol>
		<li>Dilute the secondary antibody in the blocking buffer at the chosen concentration (from 1:1, 000 to 1:50, 000), and either aliquot 100 &#181;L/well into a 96 well plate or in a box as described in section 4.4.2, and then incubate the QDB plate inside either the loaded 96 well plate or the box for 1 h at RT under constant shaking.</li>
		<li>Rinse the QDB plate gently 3 times with TBST, then wash the plate for 3 times, 5 min each with TBST under constant shaking.</li>
	</ol>
	</li>
	<li>Quantification
	<ol>
		<li>Prepare enhanced chemiluminescence substrate (ECL) by following the manufacturer&#39;s instructions.</li>
		<li>Aliquot ECL substrate into a 96 well plate (100 &#181;L/well) and insert the QDB plate inside the 96 well plate for 2 min under constant shaking.</li>
		<li>Remove the QDB plate from the 96 well plate and shake briefly to remove the excess liquid. Place the plate onto a white microtiter plate.</li>
		<li>Turn on the microplate reader, and select &#34;plate with cover&#34; on the user interface before placing the combined plates (QDB plate + white plate adaptor) inside the microplate reader for quantification. 
		Note: Make sure to use the white, non-transparent microtiter plate as an adaptor to avoid any interference from the plate. Make sure to choose &#34;plate with cover&#34; to avoid jamming the machine when combined plates (QDB plate+ 96 well plate adaptor) are placed inside the microplate reader.</li>
	</ol>
	</li>
</ol>

<ol><li>Renart, J., Reiser, J., Stark, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transfer+of+proteins+from+gels+to+diazobenzyloxymethyl-paper+and+detection+with+antisera%3A+a+method+for+studying+antibody+specificity+and+antigen+structure%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.</a> Proc Natl Acad Sci U S A. 76 (7), 3116-3120 (1979).</li>
<li>Burnette, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%22Western+blotting%22%3A+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate--polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">"Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Anal Biochem. 112 (2), 195-203 (1981).</li>
<li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets%3A+procedure+and+some+applications%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proc Natl Acad Sci U S A. 76 (9), 4350-4354 (1979).</li>
<li>Uhlen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+proposal+for+validation+of+antibodies%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">A proposal for validation of antibodies.</a> Nat Methods. 13 (10), 823-827 (2016).</li>
<li>Hawkes, R., Niday, E., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+dot-immunobinding+assay+for+monoclonal+and+other+antibodies%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">A dot-immunobinding assay for monoclonal and other antibodies.</a> Anal Biochem. 119 (1), 142-147 (1982).</li>
<li>Engvall, E., Perlmann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enzyme-linked+immunosorbent+assay+%28ELISA%29.+Quantitative+assay+of+immunoglobulin%5BTitle%5D)+AND+%22G.+Immunochemistry%22%5BJournal%5D)">Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin.</a> G. Immunochemistry. 8 (9), 871-874 (1971).</li>
<li>Engvall, E., Jonsson, K., Perlmann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enzyme-linked+immunosorbent+assay.+II.+Quantitative+assay+of+protein+antigen%2C+immunoglobulin+G%2C+by+means+of+enzyme-labelled+antigen+and+antibody-coated+tubes%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes.</a> Biochim Biophys Acta. 251 (3), 427-434 (1971).</li>
<li>Paweletz, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reverse+phase+protein+microarrays+which+capture+disease+progression+show+activation+of+pro-survival+pathways+at+the+cancer+invasion+front%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front.</a> Oncogene. 20 (16), 1981-1989 (2001).</li>
<li>Tibes, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reverse+phase+protein+array%3A+validation+of+a+novel+proteomic+technology+and+utility+for+analysis+of+primary+leukemia+specimens+and+hematopoietic+stem+cells%5BTitle%5D)+AND+%22Mol+Cancer+Ther%22%5BJournal%5D)">Reverse phase protein array: validation of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells.</a> Mol Cancer Ther. 5 (10), 2512-2521 (2006).</li>
<li>Solier, C., Langen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibody-based+proteomics+and+biomarker+research+-+current+status+and+limitations%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">Antibody-based proteomics and biomarker research - current status and limitations.</a> Proteomics. 14 (6), 774-783 (2014).</li>
<li>Tian, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+dot+blot+analysis+%28QDB%29%2C+a+versatile+high+throughput+immunoblot+method%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">Quantitative dot blot analysis (QDB), a versatile high throughput immunoblot method.</a> Oncotarget. 8 (35), 58553-58562 (2017).</li>
</ol>

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.

Among all the current available immunoassay techniques of protein analysis except ELISA, QDB analysis is the only method to achieve absolute content of a specific protein at cellular and tissue levels in a high throughput format. Although with stable isotope-labeled standard, mass spectrometry is able to achieve absolute quantification of a few proteins, this method is not yet designed for high throughput performance. In this study, we demonstrated the process of QDB analysis to achieve absolute determination of CAPG protein level in mouse tissues including kidney, spleen, and prostate. CAPG levels were found to be between 200 to 360 pg/g in mouse kidney tissues, 460 to 650 pg/g in mouse spleens, and 330 to 390 pg/g in mouse prostate tissues.
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, re-characterizing 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.

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 invention1,2,3,4,5. 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 multiplexing10.
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.

<table><tbody><tr><td>Microplate reader</td><td>Tecan</td><td>Tecan Infinite 200 PRO</td><td></td></tr><tr><td>QDB plate</td><td>Yantai Zestern Co.Ltd</td><td></td><td>Plates are available upon request for verification purpose either through email or visiting www.zestern.net.</td></tr><tr><td>Peroxidase AffiniPure Donkey Anti-Rabbit IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rat, Shp Sr Prot)</td><td>jackson</td><td>&amp;nbsp;711-035-152</td><td></td></tr><tr><td>Enhanced chemiluminesence substrate</td><td>Thermo</td><td>32134</td><td></td></tr><tr><td>Rabbit anti-CAPG&amp;nbsp;</td><td>Sino Biologic Inc</td><td>14213-T52</td><td></td></tr><tr><td>CAPG protein</td><td>Sino Biologic Inc</td><td>14213-HNAE</td><td></td></tr><tr><td>Hepes</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>Nacl</td><td>Tianjin Ruiji Jin special Chemical Co., Ltd</td><td>10461220</td><td></td></tr><tr><td>Mgcl2</td><td>Tianjin Zhiyuan Chemical Reagent Co., Ltd</td><td>20120802</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E9884-500G</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>BNN1913</td><td></td></tr><tr><td>Na2P2O7</td><td>Haituo Chinese medicine test</td><td>20160914</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T9284</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G7757</td><td></td></tr><tr><td>phenylmethylsulfonyl fluoride</td><td>Sigma</td><td>P7626-5G</td><td></td></tr><tr><td>pepstatin</td><td>Sigma</td><td>Z290033</td><td></td></tr><tr><td>leupeptin</td><td>Sigma</td><td>L2884</td><td></td></tr><tr><td>aprotinin</td><td>Sigma</td><td>A3428</td><td></td></tr><tr><td>Tris-Hcl</td><td>Sigma</td><td>T6455</td><td></td></tr><tr><td>SDS</td><td>Sigma</td><td>L5750-500G</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>43815-1G</td><td></td></tr><tr><td>Bromphenol Blue</td><td>Sigma</td><td>B-0126</td><td></td></tr><tr><td>NaF</td><td>Sigma</td><td>S7920</td><td></td></tr></tbody></table>

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.

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 &#181;g/sample for lysates from mouse kidney and spleen and 1 &#181;g/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 &#181;g for lysates from mouse spleens and kidneys, and 1 &#181;g for lysates from mouse prostates, for the final concentration of CAPG protein in these tissues as pg/&#181;g. The results are shown in Figure 1C with the original reading shown in Table 4.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56885/56885fig1.jpg" /> 
Figure 1. Representative result of a QDB analysis to determine the absolute CAPG levels in three mouse tissues (kidney, spleen and prostate). A. Examining the specificity of rabbit anti-CAPG antibody using mouse tissue lysates prepared from Spleen, Heart, Muscle, Kidney, Liver, and Prostate using western blot analysis with the negative control (IgG free BSA) and positive control (commercially available CAPG protein). B. defining the linear range of QDB analyses of rabbit anti-CAPG antibody using lysates prepared from prostate, kidney, spleen, and using purified recombinant CAPG protein standard. The results were an average of triplicates. C. Absolute determination of CAPG levels in lysates prepared from mouse kidneys, spleens, and prostates. Each bar represents one tissue from an individual mouse. The results were the average of triplicate. <a href="https://www.jove.com/files/ftp_upload/56885/56885fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 3" src="/files/ftp_upload/56885/56885table3.jpg" /> 
Table 3: Result from microplate reader of the dose studies using both serially diluted, pooled lysates from mouse spleens, kidneys, and prostates and purified recombinant CAPG protein.
<img alt="Table 4" src="/files/ftp_upload/56885/56885table4.jpg" />
Table 4: Result from microplate reader of the QDB analysis of the absolute CAPG level in mouse kidneys (8), spleens (4) and prostates (5) using a recombinant CAPG protein as standard.

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

Here we demonstrate a detailed process of quantitative dot blot analysis (QDB) by determining the absolute content of a targeted protein, capping actin protein, gelsolin-like (CAPG), in three different mouse tissues. We demonstrate a high throughput, convenient, quantitative immunoblot technique for biomarker validation at the cellular and tissue level.

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 ...

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Research

JoVE Journal

Biochemistry

11.0K Views.  Binzhou Medical University. Hier tonen we een gedetailleerd proces van kwantitatieve dot vlekkenanalyse (QDB) door de vaststelling van de absolute inhoud van een gerichte eiwit, aftopping actine eiwit, gelsolin-achtige (CAPG), in drie verschillende muis weefsels. We tonen een hoge doorvoer, handige, kwantitatieve immunoblot techniek voor biomerker validatie op het niveau van cellulaire en weefsel.

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

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and body fluids. Formalin-fixed, paraffin-embedded (FFPE) tissue sections, commonly used for long-term archiving, have emerged as valuable resources for proteomic studies. Beyond their storage benefits, researchers can isolate regions of interest (ROIs) from normal tissue regions through collaborative efforts with pathologists. Despite this potential, a streamlined approach for proteomic experiments encompassing ROI isolation, proteomic sample preparation, and MS analysis remains lacking. In this protocol, an integrated workflow that combines macrodissection of ROIs, suspension trapping-based sample preparation, and high-throughput MS analysis is presented. Through this approach, the ROIs of patients' FFPE tissues, consisting of benign serous cystic neoplasms (SCN) and precancerous intraductal papillary mucinous neoplasms (IPMN) diagnosed by pathologists, were macrodissected, collected, and analyzed, resulting in high proteome coverage. Furthermore, molecular differences between the two distinct pancreatic cystic neoplasms were successfully identified, thus demonstrating the applicability of this approach for advancing proteomic research with FFPE tissues.

Here, we establish a mass spectrometry-based proteomic method using isolated regions of interest in formalin-fixed, paraffin-embedded tissue sections. This protocol is used to analyze proteome from specific tissue areas in archived formalin-fixed, paraffin-embedded tissue sections.

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and ...

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black box&#34; because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as &#946;-actin, &#946;-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative&#160;and reliable.

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black ...

Biology

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An optimal method of quantification is accurate, has a broad linear dynamic range and maintains the structural integrity of the sample to allow for identification of individual cell types. Current methods such as immunohistochemistry (IHC), mass spectrometry, and immunoblotting each fail to meet these stipulations due to their categorical nature or need to homogenize the sample. As an alternative method, we propose the use of immunofluorescence (IF) and image analysis to determine the relative abundance of a protein of interest in FFPE tissues. Herein we demonstrate that this method is easily optimized, yields a wide dynamic range, and is linearly quantifiable as compared to the gold standard of quantitative immunoblotting. Furthermore, this method permits the maintenance of the structural integrity of the sample and allows for the distinction of various cell types, which may be crucial in diagnostic applications. Overall, this is a robust method for the relative quantification of proteins in FFPE samples and can be easily adapted to suit clinical or research needs.

We describe the use of quantitative immunoblotting to validate immunofluorescence histology coupled with image analysis as a means of quantifying a protein of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Our results demonstrate the utility of immunofluorescence histology for ascertaining the relative quantity of biomarker proteins in routine biopsy samples.

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An ...

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Presented here is an optimized high-throughput protocol developed with 16-plex tandem mass tag reagents, enabling quantitative proteome profiling of biological samples. Extensive basic pH fractionation and high-resolution LC-MS/MS mitigate ratio compression and provide deep proteome coverage.

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an ...

Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible datasets. However, despite the considerable technical advancements, sample preparation from biospecimens such as patient blood, CSF, and tissue still poses considerable challenges. For identifying biomarkers, tissue proteomics often provides an attractive sample source to translate the research findings from the bench to the clinic. It can reveal potential candidate biomarkers for early diagnosis of cancer and neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, etc. Tissue proteomics also yields a wealth of systemic information based on the abundance of proteins and helps to address interesting biological questions.
Quantitative proteomics analysis can be grouped into two broad categories: a label-based and a label-free approach. In the label-based approach, proteins or peptides are labeled using stable isotopes such as SILAC (stable isotope labeling with amino acids in cell culture) or by chemical tags such as ICAT (isotope-coded affinity tags), TMT (tandem mass tag) or iTRAQ (isobaric tag for relative and absolute quantitation). Label-based approaches have the advantage of more accurate quantitation of proteins and using isobaric labels, multiple samples can be analyzed in a single experiment. The label-free approach provides a cost-effective alternative to label-based approaches. Hundreds of patient samples belonging to a particular cohort can be analyzed and compared with other cohorts based on clinical features. Here, we have described an optimized quantitative proteomics workflow for tissue samples using label-free and label-based proteome profiling methods, which is crucial for applications in life sciences, especially biomarker discovery-based projects.

The described protocol provides an optimized quantitative proteomics analysis of tissue samples using two approaches: label-based and label free quantitation. Label-based approaches have the advantage of more accurate quantitation of proteins, while a label-free approach is more cost-effective and used to analyze hundreds of samples of a cohort.

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible ...

Quantitative Dot Blot to Estimate Target Proteins in a Tissue Lysate

Source: Qi, X., et al. <a href="https://app.jove.com/v/56885">High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB).</a> J. Vis. Exp. (2018).
This video demonstrates a quantitative dot blot, or QDB, technique for the absolute quantification of proteins of interest in a tissue sample. The proteins in the sample are immobilized on the nitrocellulose membrane inside the wells of a QDB plate. Utilizing target protein-specific antibodies labeled with a peroxidase enzyme, a chemiluminescent substrate is oxidized to produce light, which is measured to determine the protein concentration of the sample.

Source: Qi, X., et al. High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis ...

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Biological Techniques

Blot Techniques

Quantitative Western Blotting to Estimate Protein Expression Levels

Source: Huang, Y. T., et al. <a href="https://app.jove.com/v/59438">Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting.</a> J. Vis. Exp. (2019).
The video demonstrates a total protein normalization-based western blotting technique for quantifying the target protein expression in a sample. By utilizing the total protein staining of the sample, a ratiometric analysis is performed to determine the relative expression level of the target protein.

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

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