This video article describes a procedure for obtaining glycosylation profiles of 20 different glycoproteins in a hepatocellular carcinoma patient serum sample using a chemically blocked antibody microarray first glycoprotein Specific antibodies are printed directly onto microarray slides and antibody glycans are blocked to prevent lectin binding. When patient serum samples are applied, glycoproteins from the sample are captured by the antibodies. Biotinylated glycan binding proteins are then added to detect the glycans and di conjugated neu travain is added to label the biotinylated lectins.
The microarray slide is then scanned to detect fluorescence analysis of the resulting data reveals the glycosylation profiles of the sample proteins. This technique has several advantages over existing method like HPLC. This method allows for detection of individual glycoproteins in their native status.
It is faster and easier to screening glycosylation alteration for multiple proteins simultaneously. Alright, we found that that biomarkers of disease, particularly of cancer and liver cancer, which we've looked at most carefully, are often glycosylated. And the ability to detect specific glyco forms we found greatly enhances biomarkers performance in its sensitivity and in its specificity.
So this measure can provide insights into glycosylation alterations on multiple serum proteins in HCC. It can also be applied to the pathology study and biomarker discovery in other cancers and diseases such as pancreatic cancer, diabetes, and Alzheimer's disease. Demonstrating the procedure will be Chen Lu, a technician from my laboratory.
Begin this protocol by preparing the antibody microarray first in 0.8 milliliter micro centrifuge tubes on ice dilute all the antibodies that will be used for microarray printing to a concentration of 0.5 milligrams per milliliter in a minimum of 40 milliliters of PBS here, 26 different antibodies will be used as a positive control. Also prepare the same amount of 0.5 milligrams per milliliter of biotinylated BSA in PBS. Use a pipetter to aliquot 40 milliliters of each antibody into the 384 well source plate on ice.
Then in the microarray control software, designate each antibody location. Next, load the plate onto the microarray as the source. Then load 20 path microarray slides onto the microarray as target.
Set the microarray to print 48 identical subar arrays in which 27 antibodies and control proteins are spotted in triplicate in a nine by nine pattern. Start the microarray to print the antibody microarray slides. Collect the antibody microarray slides and store them in a slide cassette with desiccant.
Seal the cassette in a plastic bag to prevent protein denaturation and moisture on the slides during storage. Store the sealed microarray slides at four degrees Celsius until they are used. The single most difficult and important aspect of this procedure is the chemical blocking step.
To ensure success, it is critical that sufficient quantity of antibody are printed on the micro array light. Always prepare fresh sodium IDE and agro acid hydrocyte immediately before experiment. And be sure to carry out the oxidation procedure at four degree avoiding light.
Take the microarray slides out of the refrigerator and equilibrate them to room temperature for 30 minutes. Remove a slide from the storage box. Briefly submerge the slide in a washing basin containing PBS with 0.1%tween 20.
Then submerge the slide in 15 millimolar sodium acetate buffer with 0.1%tween for 10 minutes. Next, prepare fresh 150 millimolar sodium per iodate in 15 millimolar sodium acetate buffer, and transfer it to a slide washing basin Store in a refrigerator, avoiding light until use. Remove the slide from the sodium acetate buffer and put it into the basin containing fresh sodium puriate with the antibody side facing up.
Cover the basin with aluminum foil to avoid light. Then place the slide basin at four degrees Celsius with gentle shaking for two hours. Remove the slide from the basin and briefly rinse it in sodium acetate buffer three times for five minutes.
Incubate the slide in 300 milliliters of freshly prepared 10 millimolar hydro glutamic acid blocking solution in an incubation chamber for two hours at room temperature with gentle shaking. Remove the slides from the chamber and wash them with PBS with 0.1%tween for three minutes. Next, in a slide washing basin, incubate the microarray slide in 300 milliliters of 1%BSA in PBS with 0.5%tween for one hour at room temperature with gentle shaking.
Rinse the slides in PBS with 0.1%tween three times for three minutes. Put the slide on a slide rack and spin at 1, 200 times G for two minutes to dry the microarray slide next. To separate each subar array, a wax grid is imprinted on the slide.
After preheating the wax imprinter at 70 degrees Celsius for five minutes, load the blocked microarray slide into the wax imprinter with the antibody side facing toward the wax. Gently pull the handle to imprint wax evenly onto the slide. This method can be used to perform either glyco profiling assays for a single sample or for measuring a single glyco epitope in multiple samples.
Here we will demonstrate glyco profiling assays. Begin by diluting 40 microliters serum into 360 microliters of PBS containing 0.1%tween 0.1%bridge 35 and 100 micrograms per milliliter, each of mouse, rat, rabbit, goat, and donkey IgG. This volume is sufficient for applying six microliters of diluted serum solution onto each sub array.
Carefully apply six microliters of the diluted sample or control to each sub array of the slide. Incubate the slide in a humidified cassette with wet paper towels at room temperature for one hour. Rinse the slide with PBS with 0.1%tween three times for three minutes.
Then dry the slide by spinning it at 1200 times G for two minutes. Prepare 20 microliters of the biotinylated glycan binding proteins at 10 milligrams per milliliter in PBS tween in a 0.8 milliliter micro fuge tube on ice. Apply six microliters of the diluted biotinylated lectins to each sub array of the slide and incubate in the humidified slide box with wet paper towels at room temperature for one hour.
After rinsing the slides with PBS with 0.1%tween three times as before, dry the slide by spinning it at 1200 times G in a centrifuge for two minutes. Prepare 400 microliters of 10 milligrams per milliliter DITE 5 49 labeled neut travain in PBS 0.1%tween in a 0.8 milliliter micro fuge tube on ice. Use a repeat pipetter to apply six microliters of DITE 5 49 labeled neut travain onto each sub array and incubate the slide in the humidified slide cassette at room temperature for one hour following the incubation.
Rinse the slide with PBS 0.1%tween three times for three minutes. Dry the slide by spinning it at 1200 times G in a centrifuge for two minutes. Scan the slide by using a fluorescence microarray scanner at 10 millimeter resolution.
The laser and PMT settings should be as strong as possible while ensuring that no saturation is observed. Open the image in array Pro 3.2. Set up the array template according to the array map that shows the antibody spot locations.
Carefully align each template circle onto the corresponding spot in the image, extract the intensity of each spot into an Excel file for further analysis. An antibody micro array containing 48 identical subar arrays with 26 antibodies against 20 serum glycoproteins and biotin. BSA was designed and manufactured as described in this video to examine the importance of the blocking procedure.
In analysis of glycoprotein profiling, two identical microarray slides were generated. One was not chemically blocked while the other one was control sample was applied onto subar arrays in columns one and three and a pooled HCC serum sample was applied onto the subar arrays. In columns two and 4 22.
Biotinylated lectins specific to different glycans were then applied onto each sub array as shown in these images of the subar arrays. Lectins bound to capture antibodies in the control columns giving a high background that was indistinguishable from the serum loaded columns in the non blocked microarrays. However, when the same experiment was done on a chemically blocked antibody microarray slide, there was no or very low binding to capture antibodies in the control columns and high antigen binding in the serum loaded columns.
These results demonstrate that the chemical blocking procedure is a critical step for the measurement of glycans on antibody captured glycoproteins By following the protocol glycosylation profiles of 22 glycoproteins in HCC serum can be obtained Once mastered. This technique can be done in eight hours if it performed properly. Antibodies when modified may lose their affinities for their antigens and other properties.
So it's important to keep that in mind and use several different antibodies in different sources of antibodies Following this procedure. Other measures like detection of each protein concentration by using the same microray slide can be performed in order to answer additional questions like whether the alteration of glycosylation is due to the change of protein expression levels or solely due to the glycosylation alteration on each individual protein. Don't forget that working with pathogen containing serum samples can be extremely dangerous and precautions such as protective gloves should always be taken while performing this procedure.
