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.

Transcript

This method can be called ELISA at tissue level to distance itself from the traditional relative semi-quantitative quantification of protein content at cellular and tissue levels which is the norm in the research lab and in the clinical diagnostic field currently. The main advantages of this technique can be described in three words, high-throughput, quantitative, and absolute. If we were to add another word, it would be simple.

The implications of this technique extend into the proteomics field whereas the verification of the biomarkers can be achieved in high-throughput format. This method has broad applications throughout research and clinical field. The quantification of the protein amount at the cellular and tissue levels are still primitive at present.

Generally speaking, individuals new to this method will face no problem learning this technique and it is quite basic and require minimum lab skills to perform. We are excited to demonstrate its reliability, simplicity, and accuracy of the technique using the animal model. The samples can be prepared with any traditional method of sample preparation using any lysis buffers commonly used in the research lab with or without detergent like MP40 or Triton X.In our case, we use Triton X lysis buffer.

Place a slice of tissue of five to 100 milligrams in a 1.5 milliliter tube. Next, add 200 microliters of lysis buffer supplemented with protease and phosphatase inhibitors. Next, homogenize the tissue with a homogenizer for one minute on ice.

Then centrifuge the sample for 10 minutes at 8, 000 times g at four degrees Celsius. Collect the supernatant into new tubes and take out one microliter for measuring total protein amount in protein lysate using a protein determination kit such as a BCA assay. Incubate for 30 minutes at 37 degrees Celsius then measure the absorbance at 562 nanometers on a microplate reader.

In this step, using a recombinant protein with known concentration as a standard, dilute the protein to a concentration of 12, 000 picograms per microliter as a stock solution. Meanwhile, dilute the prepared sample lysates to four micrograms per microliter. To achieve more reliable results, we recommend using a mixture of sample lysates to avoid any difference among individual samples.

Serially dilute both the standard protein and prepared samples for a typical dose study. BSA was supplemented to ensure roughly equal amounts of the protein loaded for each sample. After that, mix 10 microliters of the serially diluted standard protein or sample lysates with 10 microliters of 2X loading buffer.

Then heat the mixture at 85 degrees Celsius for five minutes. In this procedure, place an empty pipette tip box under the QDB plate to avoid the bottom of the plate touching the table surface. Load up to two microliters of the sample to the membrane center at the bottom of an individual unit of the QDB plate.

Never allow the bottom of the QDB plate touching any surface while loading and do not load too much for individual well. In our experience, no more than four microliter per well. Next, leave the loaded QDB plate for one hour at room temperature or leave the loaded at 37 degrees Celsius for 15 minutes in a well-ventilated space.

To block the plate, dip the QDB plate in the transfer buffer and gently shake it for 10 seconds. After that, rinse the QDB plate gently with TBST three times and then wash the plate for five minutes in TBST under constant shaking. Subsequently, block the QDB plate with blocking buffer for one hour under constant shaking.

Now, dilute the primary antibody in the blocking buffer at a chosen concentration and add 100 microliters of it 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 two hours at room temperature or overnight at four degrees Celsius under constant shaking. After that, rinse the plate gently with TBST three times before it is washed with TBST three times each time for five minutes under constant shaking.

Next, dilute the secondary antibody in the blocking buffer at the chosen concentration and aliquot 100 microliters into each well of a 96-well plate. Then incubate the QDB plate inside the loaded 96-well plate for one hour at room temperature under constant shaking. Rinse the QDB plate gently three times with TBST and then wash the plate three times five minutes each with TBST under constant shaking.

For quantification, prepare ECL substrate and aliquot it into a 96-well plate at 100 microliters per well. Then insert the QDB plate inside the 96-well plate for two minutes under constant shaking. After that, remove the QDB plate from the 96-well plate and shake briefly to remove the excess liquid.

Subsequently, place the plate onto a white microtiter plate. Next, turn on the microplate reader and select plate with cover on the user interface before placing the combined plates inside the microplate reader for quantification. Make sure you choose the plate with cover to avoid mistakes.

This figure shows the specificity of rabbit anti-CAPG antibody using mouse tissue lysates prepared from spleen, heart, muscle, kidney, liver, and prostate with western blot analysis. The linear range of QDB analysis of rabbit anti-CAPG antibody using lysates prepared from prostate, kidney, spleen, and using purified recombinant CAPG protein standard was defined. The results were an average of triplicates.

And shown here is the absolute determination of CAPG levels in lysates prepared from mouse kidneys, spleens, and prostates. Once mastered, this technique can be done in four to 24 hours if it is performed properly. While attempting this procedure, it's important to remember to use a predetermined specific antibody for the whole procedure.

Following this procedure, other methods like biostatistics analysis can be performed in order to answer additional questions only achievable through absolute measurement of individual protein contents at cellular or tissue level. Since its development, this technique has paved the way for researchers in the field of proteomics research to explore the putative relationship of protein biomarkers with various diseases at population level. After watching this video, you should have a good understanding of how to perform QDB analysis in high-throughput format.

Don't forget that working with human or animal tissues can be extremely hazardous and precautions such as using gloves should always be taken while performing this procedure.

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Journal (Biochemistry)

Journal (Biochemistry)

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

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